WO2024038160A1 - Means and methods to modulate neuron excitability - Google Patents

Means and methods to modulate neuron excitability Download PDF

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WO2024038160A1
WO2024038160A1 PCT/EP2023/072727 EP2023072727W WO2024038160A1 WO 2024038160 A1 WO2024038160 A1 WO 2024038160A1 EP 2023072727 W EP2023072727 W EP 2023072727W WO 2024038160 A1 WO2024038160 A1 WO 2024038160A1
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fragment
protein
lrrc37b
fgf13a
seq
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PCT/EP2023/072727
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French (fr)
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Pierre Vanderhaeghen
Baptiste LIBÉ-PHILIPPOT
Amélie LEJEUNE
Joris DE WIT
Keimpe WIERDA
Frederic Rousseau
Joost Schymkowitz
Aikaterini KONSTANTOULEA
Nikolaos LOUROS
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Vib Vzw
Katholieke Universiteit Leuven
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Publication of WO2024038160A1 publication Critical patent/WO2024038160A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factors [FGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention relates to the field of neurological diseases. More particularly, the application discloses a novel hominid-specific pathway to modulate neuronal excitability. Means and methods are provided to treat neurological disorders characterized by affected neuron excitability. The invention further teaches methods for identification of or for screening for modulators of neuronal excitability.
  • AP action potential
  • AP occurs in an all-or-none fashion as a result of local changes in membrane potential brought about by net positive inward ion fluxes. They occur due to depolarization of the neuronal membrane, with membrane depolarization propagating down the axon to induce neurotransmitter release at the axon terminal.
  • the input (synaptic inputs) - output (AP firing rate) ratio determines neuronal excitability.
  • AIS axon initial segment
  • NAV1.6/SCN8A the voltage-gated sodium channel
  • Neuron excitability is a precisely controlled mechanism and disruption of the balance between excitation and inhibition lies at the basis of several neurological disorders. Most known examples of a hyperexcitable neuron state are seizures, epilepsy, epileptogenesis and pain. It would thus be advantageous to develop methods to modulate neuronal excitability.
  • LRRC37 family of hominid-specific genes, which encode orphan Leucine- Rich-Repeat (LRR) receptors, are expressed as a unique repertoire in human cortical neurons.
  • LRR Leucine- Rich-Repeat
  • LRRC37B protein is a receptor displayed selectively at the level of the axon initial segment (AIS) of human cortical pyramidal neurons. It was surprisingly found that LRRC37B overexpression in vivo lead to reduced intrinsic neuron excitability.
  • AIS axon initial segment
  • LRRC37B was found to act through direct binding to secreted FGF13A ligand, leading to inhibition of the voltagegated sodium channel a (alpha) subunit named NAVI.6 (encoded by the SCN8A gene), the main voltagegated sodium channel responsible to AP generation.
  • Said FGF13A ligand is referred to as FGF13A protein or FGF13A, in the description below.
  • LRRC37B also binds to the regulatory SCN1B beta-subunit of the PVDH/LRRC37/769 voltage-gated sodium channel, and thereby negatively regulates SCN1B interaction with the alpha subunit, known as NAVI.6.
  • LRRC37B Ex vivo physiological experiments on adult human cortical tissue revealed reduced excitability in pyramidal neurons expressing LRRC37B, indicating its physiological relevance for human neuronal function.
  • the data presented herein identify LRRC37B as a human-specific modifier of cortical neuron excitability that acts through interactions with FGF13A and/or SCNB1, preferably acts through interactions with FGF13A and SCNB1.
  • the novel modulators and/or modulation mechanisms disclosed herein with important implications, may allow for better understanding of brain function, evolution, and/or provide for new agents, means and/or methods for use in excitability disorders such as seizures and epilepsy.
  • a modulator of the LRRC37B-FGF13A-SCN1B complex for use as a medicine, more particularly for modulating neuronal excitability, even more particularly for treating neuronal excitability disorders such as seizures and epilepsy.
  • a modulator of the interaction between the LRRC37B protein or receptor and the NAVI.6 voltage-gated sodium channel for use as a medicine is provided.
  • said modulator is a modulator of the LRRC37B-FGF13A interaction and/or of the LRRC37B-SCN1B interaction, preferably LRRC37B-FGF13A interaction or of the LRRC37B-SCN1B interaction.
  • the application also provides an isolated FGF13A protein fragment, preferably said isolated FGF13A fragment comprising or consisting of an amino acid sequence as depicted in SEQ ID No. 4, more preferably said isolated FGF13A fragment comprising or consisting of an amino acid sequence SEQ ID No. 15 for therapeutic application and/or for screening purposes.
  • the application also provides the isolated LRRC37B protein fragment comprising or consisting of the amino acid sequence as depicted in SEQ ID No. 8 or in SEQ ID No. 12 for therapeutic applications or and/or for screening purposes.
  • the application also provides a modulator of the interaction between LRRC37B and the NAVI.6 voltagegated sodium channel for use as a medicine.
  • said modulator is a peptide comprising a fragment of FGF13A, said fragment of FGF13A preferably being FGF13A S-fragment as depicted in SEQ ID No. 4 or a peptidomimetic thereof.
  • said modulator is a peptide comprising the fragment of FGF13A comprising or consisting of SEQ ID No. 15 or a peptidomimetic thereof.
  • the application provides methods of identifying modulators of neuronal excitability, more particularly modulators of the LRRC37B-FGF13A-SCN1B complex, even more particularly modulators of the LRRC37B-FGF13A interaction and/or of the LRRC37B-SCN1B interaction, preferably modulators of the LRRC37B-FGF13A interaction or of the LRRC37B-SCN1B interaction.
  • Said methods comprise the steps of contacting the LRRC37B protein or a fragment thereof with the FGF13A protein or a fragment thereof and/or the SCN1B protein or fragment thereof in the presence or absence of a test compound and identifying said test compound as modulator of neuronal excitability and function if said test compound is statistically significantly reducing or increasing the binding of the LRRC37B protein or fragment thereof with the FGF13A protein and/or with the SCN1B protein or fragments thereof, compared to identical conditions but in absence of the test compound.
  • the binding between the protein partners can be determined, for example, by immunologic or radiologic detection, co-sedimentation, co-immunoprecipitation or electron microscopy.
  • the LRRC37B or the fragment thereof can be provided by cells expressing LRCC37B or the fragment thereof.
  • the FGF13A or the fragment thereof can be administered extracellularly to the cells expressing LRRC37B or the fragment thereof.
  • said test compound is a peptide consisting of less than 20 contiguous amino acids and is a fragment of the LRRC37B-specific domain as, for example, depicted in SEQ ID No. 12.
  • said test compound is, for example, a peptidomimetic of said peptide.
  • the application also provides a method of identifying a modulator of neuron excitability comprising the steps of generating a peptide consisting of less than 20 contiguous amino acids wherein the peptide is a fragment of the FGF13A S-fragment, herein also referred to as FGF13A S-domain as depicted in SEQ ID No. 4 or SEQ ID No. 15 or generating a peptidomimetic of said peptide, and contacting one or more of the peptides or peptidomimetics with the NAVI.6 voltage-gated sodium channel as depicted in SEQ ID No.
  • NAVI.6 or the functional fragment thereof is provided by cells expressing NAVI.6 or the functional fragment thereof.
  • said cells are electrically excitable cells, such as neurons or cardiac cells.
  • Figure 1A shows the LRRC37 transcripts detected in the cerebral cortex in mouse, macaque and human.
  • RPKM stands for reads per kilobase million.
  • Figures 1B-C illustrate the proportion of LRRC37 positive cell types (B) and pyramidal neurons (C) in human postnatal cortical samples at different ages (yo: years old). RPKM stands for reads per kilobase million.
  • Figure ID shows the expression of LRRC37 transcripts in cell types of human and chimpanzee prefrontal cerebral cortex.
  • CPM stands for counts per million.
  • Figure IE illustrates the expression of LRRC37 transcript in CPM during the postnatal human life (from neonates to adulthood) in the prefrontal cerebral cortex.
  • Figure 2A-B show that LRRC37B colocalizes with ankyrin-G in a subset of human neurons from individuals older than 2yo with no differences in the AIS length of LRRC37B+ neurons compared to LRRC37B- neurons (Mann-Whitney test).
  • Figure 2C shows the immunodetection of LRRC37B in the mouse cerebral cortex transfected with LRRC37B cDNA and colocalization with ankyrin-G.
  • Figure 2Da-b and E show that LRRC37B mouse transfected neurons have a decreased neuronal excitability compared to control neurons transfected with EGFP cDNA only (control): the LRRC37B positive cells display a decreased action potential (AP) firing rate (2-way ANOVA test) (D a-b), an increased rheobase (E, left) and an increase AP risetime (E, center) and width (E, right) (Mann-Whitney test).
  • Figure 2F illustrates that LRRC37B is detected in a subset of human cortical AIS from individuals older than 2 year old to reach a plateau from 10 years old. ns, non-significative; *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001.
  • FIG. 3 herein below, the FGF13A S-fragment as depicted in SEQ ID No. 4 is referred to as "ExonS".
  • Figure 3A is an illustration of the several FGF13 spliced isoforms (upper panel) and shows that FGF13A is the only secreted FGF13 (lower panel).
  • Figure 3B shows that LRRC37B binds only to FGF13A as demonstrated by LRRC37B-HA immunoprecipitation from co-transfected HEK-293 cells with cDNAs coding for FGF13A, FGF13B, FGF13VY and the core domain of FGF13 proteins.
  • Figure 3C shows the coimmunoprecipitation with LRRC37B-HA of recombinant FGF13A and its synthetic ExonS applied in the culture medium of HEK-293 cells transfected with LRRC37B-HA cDNA.
  • Figure 3D shows that FGF13A co- immunoprecipitates in transfected HEK-293 cells with the LRRC37B extracellular part (LRRC37Bectoml) as well as with its leucin-rich repeats (LRR) (LRRC37Bectom2) but not with the extracellular part devoid of the LRR (LRRC37Becto).
  • Figure 3E is an illustration of the different LRRC37B protein fragments tested.
  • Figure 3F depicts a peptide array experiment with LRR protein application on 10 amino acid peptides windows covering the ExonS sequence which enables to identify two possible binding sites: ExonS amino acids 4-23 (as depicted in SEQ ID No. 16, and herein referred to as “ExonS 4-23”) and ExonS amino acids 40-63 (as depicted in SEQ ID No. 15, and herein referred to as “ExonS 40-63”).
  • Figure 3G shows fluorescent polarization (FP) assay between LRR protein (different doses) and ExonS of SEQ ID No. 4 (marked as "ExonS”), and also fragments of ExonS: ExonS 40-63, depicted in SEQ ID No.
  • FP fluorescent polarization
  • Exon S 40-63 Exon S 40-63
  • ExonU ExonU
  • FGF13B ExonU two Random peptides as defined in Figure 3F
  • LRR binds to the ExonS at an affinity of 530nM
  • ExonS 40-63 i.e. the peptide of a sequence as depicted in SEQ ID No. 15
  • Figure 3H shows affinity estimation binding assays with peptide application of ExonS or fragments thereof (the fragment as depicted in SEQ ID No. 16, herein annotated as "ExonS 4-23"; the fragment as depicted in SEQ ID No.
  • ExonS 40-63 or the FGF13A S-fragment as depicted in SEQ ID No. 4, herein annotated as “ExonS” on HEK-293T cells transfected for LRRC37B_ECTO (as defined in panel D-E) or empty vector: only ExonS 40-63 (affinity 0.4nM) and ExonS (affinity 4nM) bind to LRRC37B.
  • ExonS the FGF13A S-fragment as depicted in SEQ. ID No. 4 is referred to as “ExonS”
  • shorter peptide fragment consisting of amino acids from position 40 to 63 as depicted in Seq ID No.
  • ExampleS 40-63 15 is referred to as "ExonS 40-63".
  • Figure 4A(a)-B(a) show that extracellularly applied recombinant FGF13A on mouse cortical sections decreases neuronal excitability (A), AP firing rate (B left, 2-way ANOVA test and paired Wilcoxon test) and rheobase (B right, paired Wilcoxon test).
  • Figure 4A(b) shows that extracellularly applied recombinant FGF13A, synthetic ExonS, ExonS 40-63 on mouse cortical sections decreases neuronal excitability when compared to ExonS 1-39 (consisting of aa from position 1 to position 39 of ExonS sequence).
  • Figure 4B(b) shows normalization (to values before application for each neuron) of AP firing rate (left, paired Wilcoxon tests) and rheobase (right, paired Wilcoxon test).
  • Figure 4C(a) shows that extracellular application of the FGF13A S fragment on mouse cortical sections is sufficient to decrease neuronal excitability (left) in contrast to intracellular application of said FGF13A S fragment (right).
  • Figure 4C(b) shows that intracellular application of FGF13A does not lead to decreased neuronal excitability (right, Mann-Whitney test).
  • Figure D(a)-G(a) shows the effect of extracellular application of FGF13A (D(a)-E(a)) and the FGF13A S fragment (F(a)-G(a)) on Na (D(a)-F(a)) and K (E(a)- G(a)) currents.
  • Figure 4D(b)-E(b) shows the effect of extracellular application of FGF13A, ExonS, ExonS 40-63 but not ExonS 1-39 on Na (D(b), right normalization to values before application for each neuron, paired Wilcoxon tests) and K currents (E(b), right normalization to values before application for each neuron, paired Wilcoxon tests).
  • Figure 4H shows the NAVI.6 immunoprecipitations from HEK-293 cells transfected for NAVI.6 and FGF13A with or without LRRC37B, more particularly NAVI.6 binds FGF13A and that they form a complex with LRRC37B.
  • Figure 41 shows the co-immunoprecipitation of FGF13A with LRRC37B-HA from cortical extracts (P17) of LRRC37B-HA/EGFP transfected mice compared to control.
  • Figures 4J-M show that LRRC37B co-localizes with FGF13A and NAVa subunits based on STED microscopy of the AIS of mouse neurons transfected for LRRC37B (J) and that FGF13A abundance increases (K) at the AIS (L) upon LRRC37B expression in contrast to NAVa subunits (M).
  • Figure 5A shows the SCN1B immunoprecipitations from HEK-293 cells transfected for SCN1B +/-, LRRC37B +/- and FGF13A +/- cDNAs.
  • Figure 5B is an illustration of the LRRC37B deletion mutants.
  • Figure 5C shows that SCNB1 co-immunoprecipitates with the LRRC37B extracellular part as well as its specific LRRC37B domain but not with the extracellular part devoid of the specific B domain in transfected HEK- 293 cells.
  • Figure 5D shows the NAVI.6 immunoprecipitations from HEK-293 cells transfected for NAVI.6, SCN1B and LRRC37B +/- cDNAs.
  • Figure 5E shows the immunoprecipitation of the extracellular part of SCN1B with a synthetic peptide corresponding to the LRRC37B specific domain (LB133-186) from HEK- 293 cells transfected for the cDNA coding for the extracellular part of SCN1B.
  • Figure 5F shows the SCNB1 and NAVI.6 immunoprecipitations from HEK-293 cells transfected for NAVI.6, SCN1B, with or without LB133-186.
  • Figure 6A(a) shows that LRRC37B forms a complex with FGF13A and SCN1B in the human cortex.
  • Figure 6A(b) shows that LRRC37B forms a complex with FGF13A, SCN1B and NAVa subunits (i.e. NAV 1.6) in the human cortex.
  • Figures 6B(a)-E(a) show that LRRC37B+ neurons display a decreased excitability compared to LRRC37B- neurons (2-way ANOVA test for the AP firing rate, Mann-Whitney test for other parameters). *, p ⁇ 0.05; ****, p ⁇ 0.0001.
  • Figures 6B(b) and F show that LRRC37B+ neurons display a decreased excitability compared to LRRC37B- neurons (Mann-Whitney tests). Legend: ns, nonsignificant; *, p ⁇ 0.05; ***, p ⁇ 0.001; ****, p ⁇ 0.0001.
  • Figure 7 is an illustration showing the proposed model on how the LRRC37B-FGF13A-SCN1B complex acts on the NAVI.6 voltage-gate sodium channel to modulate neuron excitability in the human cortex.
  • the term "about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). For example, if it is stated that a modulator of the LRRC37B-FGF13A-SCN1B interaction reduces the activity or function of NAVI.6 in a cell by at least about 60%, it is implied that the NAVI.6 activity or function is reduced by a range of 50% to 70%.
  • nucleic acid As used herein, the terms “nucleic acid”, “nucleic acid sequence” or “nucleic acid molecule” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acids may have any three-dimensional structure, and may perform any function, known or unknown.
  • Non-limiting examples of nucleic acids include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers.
  • the nucleic acid molecule may be linear or circular.
  • the nucleic acid may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5' or 3' untranslated regions, a reporter gene, a selectable marker or the like.
  • the nucleic acid may comprise single stranded or double stranded DNA or RNA.
  • the nucleic acid may comprise modified bases or a modified backbone.
  • a nucleic acid that is up to about 100 nucleotides in length, is often also referred to as an oligonucleotide.
  • "Nucleotides" as used herein refer to the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention include both naturally occurring and non-naturally occurring nucleotides.
  • nucleotides, such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which are absent in nucleosides).
  • nucleoside A nucleotide without a phosphate group is called a "nucleoside” and is thus a compound comprising a nucleobase moiety and a sugar moiety.
  • nucleobase means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid.
  • Naturally occurring nucleobases of RNA or DNA comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • amino acids refer to the structural units (monomers) that make up proteins. They join together to form short polymer chains called peptides or longer chains called either polypeptides or proteins. These chains are linear and unbranched, with each amino acid residue within the chain attached to two neighbouring amino acids. Twenty amino acids encoded by the universal genetic code are naturally incorporated into polypeptides and are called proteinogenic or natural amino acids.
  • Natural amino acids or naturally occurring amino acids are glycine (Gly or G), Alanine (Ala or A), Valine (Vai or V), Leucine (Leu or L), Isoleucine (He or I), Methionine (Met or M), Proline (Pro or P), Phenylalanine (Phe or F), Tryptophan (Trp or W), Serine (Ser or S), Threonine (Thr or T), Asparagine (Asn or N), Glutamine (Gin or Q), Tyrosine (Tyr or Y), Cysteine (Cys or C), Lysine (Lys or K), Arginine (Arg or R), Histidine (His or H), Aspartic Acid (Asp or D) and Glutamic Acid (Glu or E).
  • nucleic acid or amino acid sequences refer to two or more sequences that are the same or have a specified percentage of nucleotides or amino acid residues respectively that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • the percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of nucleotide or amino acid sequences.
  • percent sequence identity or “% sequence identity” or “percent identity” or “% identity” between two polynucleotide or polypeptide sequences refers to the number of identical matched positions shared by the sequences over a comparison window, taking into account additions or deletions (i.e. gaps) that must be introduced for optimal alignment of the two sequences.
  • a matched position is any position where an identical nucleotide or amino acid is presented in both the target and reference sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides or amino acids. Likewise, gaps presented in the reference sequence are not counted since target sequence nucleotides or amino acids are counted, not nucleotides or amino acids from the reference sequence.
  • sequence alignment algorithm is the algorithm described in Karlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified in Karlin et a!., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, and incorporated into the NBLAST and XBLAST programs (Altschul et a!., 1991, Nucleic Acids Res., 25:3389-3402).
  • Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.
  • BLAST-2 Altschul et al., 1996, Methods in Enzymology, 266:460-480
  • ALIGN ALIGN-2
  • Megalign Megalign
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6).
  • the GAP program in the GCG software package which incorporates the algorithm of Needleman and Wunsch (J.
  • Mol. Biol. (48):444-453 (1970)) can be used to determine the percent identity between two amino acid sequences (e.g., using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5).
  • the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)).
  • the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM 120 with residue table, a gap length penalty of 12 and a gap penalty of 4.
  • One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain aspects, the default parameters of the alignment software are used.
  • sequence alignments are not limited to binary sequence-sequence comparisons exclusively driven by primary sequence data. Sequence alignments can be derived from multiple sequence alignments.
  • One suitable program to generate multiple sequence alignments is ClustalW2, available from www.clustal.org.
  • Another suitable program is MUSCLE, available from www.drive5.com/muscle/.
  • ClustalW2 and MUSCLE are alternatively available, e.g., from the EBI (European Bioinformatics Institute).
  • the percentage identity "X" of a first nucleotide sequence to a second nucleotide sequence is calculated as 100 x (Y/Z), where Y is the number of nucleotide residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence. Different regions within a single polynucleotide target sequence that align with a polynucleotide reference sequence can each have their own percent sequence identity.
  • percent sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.
  • the degree of identity, between a given reference nucleotide sequence and a nucleotide sequence which is a homologue of said given nucleotide sequence will preferably be at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the degree of identity is given preferably for a nucleic acid region which is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the entire length of the reference nucleic acid sequence.
  • the degree of identity is given preferably for at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or 200 nucleotides, preferably contiguous nucleotides.
  • the degree/percentage of similarity or identity is given for the entire length of the reference nucleic acid sequence.
  • amino acid identity refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin,
  • the degree of identity, between a given reference amino acid sequence and an amino acid sequence which is a homologue of said given amino acid sequence will preferably be at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the degree of identity is given preferably for an amino acid region which is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the entire length of the reference amino acid sequence.
  • the degree of identity is given preferably for at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or 200 amino acids, preferably contiguous amino acids.
  • the degree/percentage of similarity or identity is given for the entire length of the reference amino acid sequence.
  • Homologue or “homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
  • Contiguous as used herein means next or together in sequence, hence the contiguous nucleotides or amino acids are linked nucleotides or amino acids (i.e. no additional nucleotides or amino acids are present between those that are linked).
  • SEQ ID No. X refers to a biological sequence consisting of the sequence of amino acids or nucleotides given in the SEQ ID No. X.
  • a protein defined in/by SEQ ID No. X consists of the amino acid sequence given in SEQ ID No. X.
  • a further example is an amino acid sequence comprising SEQ ID No. X, which refers to an amino acid sequence longer than the amino acid sequence given in SEQ ID No. X but entirely comprising the amino acid sequence given in SEQ ID No. X (wherein the amino acid sequence given in SEQ ID No. X can be, for example, located N-terminally or C-terminally in the longer amino acid sequence, or can be embedded in the longer amino acid sequence), or to an amino acid sequence consisting of the amino acid sequence given in SEQ ID No. X.
  • Compound or “test compound” used in the screening methods of the present application is not limited to a specific type of a compound and means any chemical or biological compound, including simple or complex organic and inorganic molecules, peptides, peptidomimetics, proteins, antibodies, carbohydrates, nucleic acids or derivatives thereof.
  • the term “compound” is used herein in the context of a "drug candidate compound” or a “candidate compound for lead optimization” in therapeutics, described as identified with the screening methods herein disclosed.
  • these compounds comprise organic or inorganic compounds, derived synthetically or from natural resources.
  • the compounds include polynucleotides, lipids or hormone analogs that are characterized by low molecular weights.
  • Other biopolymeric organic test compounds include small peptides or peptide-like molecules (peptidomimetics) comprising from about 2 to about 40 amino acids and larger polypeptides comprising from about 40 to about 500 amino acids, such as antibodies or antibody conjugates.
  • small molecule compound refers to a low molecular weight (e.g., ⁇ 900 Da or 40 ⁇ 500 Da) organic compound.
  • the compounds also include polynucleotides, lipids or hormone analogues that are characterized by low molecular weights.
  • Other biopolymeric organic test compounds include small peptides or peptide-like molecules (peptidomimetics) comprising from about 2 to about 40 amino acids and larger polypeptides comprising from about 40 to about 500 amino acids, such as antibodies or antibody conjugates.
  • Compound libraries are a large collection of stored compounds utilized for high throughput screening. Compounds in a compound library can have no relation to one another, or alternatively have a common characteristic. For example, a hypothetical compound library may contain all known compounds known to bind to a specific binding region. As would be understood by one skilled in the art, the methods of the application are not limited to the types of compound libraries screened. For high-content screening, compound libraries may be used. Examples include, but are not limited to, natural compound libraries, allosteric compound libraries, peptide libraries, antibody fragment libraries, synthetic compound libraries, etc.
  • high throughput screening methods involve providing a library containing a large number of compounds (candidate compounds) potentially having the desired activity.
  • Such "combinatorial chemical libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity.
  • the compounds identified can serve as conventional "hit compounds” or can themselves be used as potential or actual therapeutics.
  • Test compounds can be selected from a collection of compounds, more particularly a library of biological and/or chemical compounds.
  • Nonlimiting examples of said library can be a library comprising small molecules, compounds with known functions, FDA approved drugs, compounds pre-screened on bioactivity or can be a drug repurposing library.
  • the screening methods herein described are "high content screening” (HCS) methods.
  • HCS high content screening
  • the present invention is not limited to the speed or automation of the screening process. The method is neither limited to large or high-throughput or any scale, and can be refined based on the availability of test compounds or other variable features of the screening assay.
  • the term "vector” refers to any linear or circular DNA construct comprising one of the nucleic acid molecules of the application.
  • the vector can refer to an expression cassette or any recombinant expression system for the purpose of expressing one of the nucleic acid molecules of the application in vitro or in vivo, constitutively or inducibly, in any cell, including mammalian cells.
  • the vector can remain episomal or integrate into the host cell genome.
  • the vector can have the ability to self-replicate or not (i.e. drive only transient expression in a cell).
  • the term includes recombinant expression cassettes that contain only the minimum elements needed fortranscription of the recombinant nucleic acid.
  • the vector of the invention can a "recombinant vector" which is by definition a man-made vector.
  • the vector can also be a viral vector including lentiviral, retroviral, adenoviral and adeno-associated viral vectors.
  • Neuron cells are a type of cell in the central nervous system, which receive, integrate, and pass along information by releasing neurotransmitters. Said neurotransmitters are chemicals that cross-over from the terminal button at the end of an axon over the synapse to the neighbouring neuron.
  • Non-limiting examples of neuron cells are primary cortical neurons, primary basal forebrain cholinergic neurons, primary neural stem cells, sensory neurons (e.g. retinal cells, olfactory epithelium cells), motor neurons (e.g. spinal motor neurons, pyramidal neurons, Purkinje cells) and interneurons (e.g. dorsal root ganglia cells).
  • the "action potential" as used herein is a rapid and reversible reversal of the electrical potential difference across the plasma membrane of excitable cells such as neurons, muscle cells and some endocrine cells.
  • the membrane potential rapidly changes from its resting level of approximately -70 mV to around +50 mV and, subsequently, rapidly returns to the resting level again.
  • the neuronal action potential forms an important basis for information processing, propagation and transmission. In muscle cells, the action potential precedes, and is necessary to bring about, muscle contraction. Some endocrine cells also exhibit action potentials, where the excitation leads to hormone secretion.
  • Treatment refers to any rate of reduction or retardation of the progress of the disease or disorder compared to the progress or expected progress of the disease or disorder when left untreated. More desirable, the treatment results in no or zero progress of the disease or disorder (i.e. "inhibition” or “inhibition of progression") or even in any rate of regression of the already developed disease or disorder.
  • Reduction or “reducing” as used herein refers to a statistically significant reduction, more particularly said statistically significant reduction is an at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% reduction compared to the control situation.
  • Statistical significance plays a pivotal role in statistical hypothesis testing. It is used to determine whether the null hypothesis should be rejected or retained.
  • the null hypothesis is the default assumption that nothing happened or changed, hence that there is no difference for example in the activity of the NAVI.6 channel in the presence of a test compound compared to the activity of the NAVI.6 channel in the absence of said test compound.
  • an observed result has to be statistically significant, i.e. the observed p-value is less than the pre-specified significance level a.
  • the p-value of a result, p is the probability of obtaining a result at least as extreme, given that the null hypothesis were true.
  • a is 0.05.
  • a is 0.01.
  • a is 0.001.
  • an isolated FGF13A_S peptide as disclosed herein refers to a FGF13A derived peptide, which has been purified from the molecules which flank it in a naturally- occurring state, e.g. the C-terminal part of FGF13A has been removed.
  • An isolated peptide can be generated by amino acid chemical synthesis or can be generated by recombinant production.
  • Another example concerns an isolated neuronal cell, which refers to a neuronal cell which has been extracted and purified from the naturally-occurring state, involving tissue.
  • An isolated neuronal cell preparation can be obtained from several neuronal tissue types using for example specialized commercial kits that make use of proteases to digest intercellular protein junctions followed by gentle mechanical disruption to liberate individual cells, or for instance but not limited to the exemplified method.
  • LRRC37B-FGF13A-SCN1B complex as mentioned herein, it is meant that LRRC37B, FGF13A and SCN1B can be co-immunoprecipitated together, for example by transfection in HEK cells and from human tissue.
  • a complex it is meant that components of said complex can be coimmunocoprecipitated together, for example, NAVa subunits, LRRC37B, FGF13A and SCN1B can be co- immunoprecipitated together (human cortical biopsy), LRRC37B, FGF13A and NAVI.6 can be co- immunoprecipitated together (transfection in HEK cells).
  • FGF13A S-fragment or S-domain also referred to as "ExonS"
  • FGF13A S-fragment sequence being depicted in SEQ ID No. 4.
  • homologue of FGF13A S-fragment it is meant a protein or a peptide comprising at least 80% 81% 82% 83% 84% 85% 86°% 87% 88% 89% 90% 91% 92% 93% 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the FGF13A protein fragment depicted in SEQ ID No. 4, wherein the homologue has the same or the similar function or effect as the GFG13A protein fragment as depicted in SEQ ID No. 4.
  • FGF13A protein fragment also referred to as "FGF13A fragment", as used herein, it is meant any protein or peptide fragment having at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% amino acid sequence identity with the FGF13A protein as depicted in SEQ ID No. 2.
  • homologue of said FGF13A fragment it is meant a protein or a peptide comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with said FGF13A fragment, wherein the homologue has the same or the similar function or effect as the GFG13A fragment.
  • LRRC37B is an axon initial segment (AIS) hominid-specific protein sufficient to modulate neuronal excitability.
  • the AIS is a unique subcellular compartment at the beginning of the axon.
  • AIS proteins organize as a layered scaffold spanning from microtubules to the plasma membrane (Leterrier 2016 Curr Top Membr 77: 185- 233).
  • the central component of this scaffold is ankyrin G (ANK3) that concentrates at the AIS and anchors AIS-specific membrane proteins, including voltage-gated sodium (NAV) and potassium (Kv) channels.
  • NAV voltage-gated sodium
  • Kv potassium
  • Voltage-gated sodium channels primarily function to provide an explosive, regenerative inward current during the rising phase of the action potential (AP) (Clark et al 2009 Neuroscientist 15:651-668; Kole and Stuart 2012 Neuron 73:235-247). At normal resting potentials, VGSCs will be in a closed confirmation and be non-conducting. VGSCs generally adopt an open conformation upon membrane depolarization (about -40mV) to allow a flow of sodium ions down a concentration gradient from the outside of the cell to the inside of the cell.
  • VGSCs are formed by an a subunit which constitutes the channel itself and beta subunits (SCN1-2-3-4B) which modulate its gate (Catterall et al 2005 Pharmacol Rev 57: 411-25).
  • the a subunits (NAVa subunits) are further classified into nine subtypes (NAVI.1-1.9), the expression of which is both cell- and tissue-specific (Catterall et al 2005 Pharmacol Rev 57: 411-25).
  • the main subtypes expressed in the brain are the NAVI.6 (coded by SCN8A) localized in the axonal initial segment, nodes of Ranvier, cell somas and dendrites; NAV1.1 is found in the cell soma; and NAVI.2 is present in unmyelinated axons and myelinated axons during development (Mantegazza et al 2010 Lancet Neurol 9: 413-424). It is generally accepted that abnormal expression of VGSCs contribute to the changes in intrinsic neuronal excitability that underlie several neurological disorders including epilepsy and migraine (Mantegazza et al 2010 Lancet Neurol 9: 413-424).
  • NAV1.1, NAVI.2, NAVI.3, NAVI.6, as well as beta subunits have been shown in animal models of epilepsy and in human brain tissue in acquired epilepsy (Aronica et al 2001 Eur J Neurosci 13: 1261-1266; Blumenfeld et al 2009 Epilepsia 50: 44-55; Ketelaars et al 2001 Neuroscience 105: 109-120; Klein et al 2004 Brain Res 1000: 102-109).
  • LRRC37B concentrates inhibitory effects of FGF13A on the channel function of voltage-gated sodium channel, thereby reducing neuronal excitability, specifically at the AIS level.
  • the binding between FGF13A and NAVI.6 inhibits NAVI.6 action.
  • NAVI.6 and “NAVI.6 voltage-gated sodium channels” are used as synonyms throughout the description of the present invention.
  • LRRC37B or FGF13A overexpression as well as extracellular administration of the FGF13A fragment binding to LRRC37B decreases neuron excitability.
  • SCN1B is a NAV p-subunit that modulates VGSCs, in particular NAVI.6 (Wimmer et al 2010 J Clin Invest 120: 2661-2671).
  • the NAV -subunit family consists of four proteins: pi-4, coded by genes SCN1B to SCN4B, respectively. These are single-span transmembrane proteins oriented with the amino terminus facing the extracellular space. The extracellular domain presents a conserved immunoglobulin domain, homologous to the one in cell adhesion molecules.
  • the carboxyl terminus associates with cytoskeletal and scaffolding proteins, pi (or SCN1B) and P2 associate with ankyrin-G and ankyrin-B in both brain and heart, and their interaction is critical for channel surface expression and modulates the channel function in vivo (Cerrone et al 2014 Cardiac Electrophysiology).
  • P-subunits function in concert with a-subunits to promote channel trafficking to the plasma membrane and to modulate the NAV biophysical properties (Deschenes 2018 Cardiac Electrophysiology). While there are some discrepancies in some of the observations, one consensus is with the increase in current density seen when expressing SCN1B with the different NAVa isoforms. Coexpression of SCN1B subunits with NAVI.5 in vitro leads to increased a-subunit expression at the plasma membrane resulting in an augmentation in current density (Nuss et al 1995 J Gen Physiol 106: 1171-1191; Qu et al 1995 J Biol Chem 270: 25696-25701; Isom et al 1995 J Biol Chem 270: 3306-3312).
  • mouse neurons that obviously lack the herein disclosed hominid-specific regulation of neuron excitability are less excitable compared to human neurons (Beaulieu-Laroche et al 2018 Cell 175: 643-651. el4; Beaulieu-Laroche et al 2021 Physiol Rev 99: 1079-1151). This makes the LRRC37B- dependent regulation of particular interest to understand the molecular basis of human neuronal evolution.
  • LRRC37B can affect information processing through neuronal gain modulation, by which neurons adapt to changing inputs (Ferguson and Cardin 2020 Nat Rev Neurosci 21: 80-92). Neuronal gain modulation relies largely on the regulation of synaptic inputs, but also output modulation, including at the level of the AIS (Debanne et al 2019 Curr Opin Neurobiol 54: 73-82). Moreover, neuronal plasticity and learning involves modulation of the neuronal gain including at the level of the output (Ferguson and Cardin 2020 Nat Rev Neurosci 21: 80-92; Jamann et al 2021 Nat Commun 12: 1-14.). The modulation of neuronal excitability by LRRC37B could thus influence sensory processing and plasticity, at least in part through gain modulation, and thereby contribute to human-specific properties of cortical circuits.
  • LRRC37B binds to FGF13A and to SCN1B, which are both mutated in severe forms of epilepsy (Devinsky et al 2018 Epilepsy Nat Rev Dis Prim 4: 18024; Wimmer et al 2010 Physiol 588: 1829-1840).
  • FGF13A peptide administration and/or administration of the B peptide encoding the B-specific domain of LRRC37B is of high relevance in modulating neuronal excitability and for treating epilepsy and seizures in general.
  • a modulator of the interaction between a LRRC37B protein and a NAVI.6 voltage-gated sodium channel in particular a modulator of the LRRC37B-FGF13A-SCN1B, the LRRC37B- FGF13A, the FGF13A-NAV1.6, the SCN1B-NAV1.6, the LRRC37B-SCN1B or the LRRC37B-FGF13A-SCN1B- NAV1.6 interactions is provided for use as a medicament.
  • a modulator of the LRRC37B protein and NAVI.6 voltage-gated sodium channel functions for use as a medicine is provided.
  • a method of treatment comprising the step of administering to a subject a modulator of the LRRC37B-FGF13A-SCN1B, LRRC37B-FGF13A, FGF13A-NAV1.6, SCN1B- NAV1.6, LRRC37B-SCN1B and/or the LRRC37B-FGF13A-SCN1B-NAV1.6 interactions.
  • the modulator is provided for modulating neuron excitability and/or function.
  • Several neurological disorders are known that are caused by an aberrant modulation of neuron excitability.
  • Nonlimiting examples are seizures, epilepsy, epileptogenesis, neuropathic pain, anxiety, depression, Alzheimer's disease, cognitive impairments, dystonia, narcolepsy and spasticity.
  • the modulator of the LRRC37B-FGF13A-SCN1B, LRRC37B-FGF13A, FGF13A-NAV1.6, SCN1B-NAV1.6, LRRC37B-SCN1B and/or the LRRC37B-FGF13A-SCN1B-NAV1.6 interactions is provided for use to treat seizures, epilepsy or epileptogenesis.
  • “Seizures” as used herein refers to paroxysmal alteration of neurologic function caused by the excessive, hypersynchronous discharge of neurons in the brain (Stafstrom & Carmant 2015 Cold Spring Harb Perspect Med 5(6): a022426). They can be generalized (leading to absences, tonic-clonic, myoclonic or atonic manifestations) or focal (manifestations are linked to the brain area affected) (Stafstrom & Carmant 2015 Cold Spring Harb Perspect Med 5(6): a022426). Based on their etiology, seizures are also classified into symptomatic (lesion, syndrome) or idiopathic seizures.
  • Epilepsy is a chronic non-communicable disease of the brain that affects around 50 million people worldwide. It is characterized by recurrent seizures.
  • Epileptogenesis is the process whereby a previously normal brain is functionally altered and biased towards the generation of the abnormal paroxysmal electrical activity that defines chronic seizures.
  • the modulator statistically significantly improves, stimulates or enhances the interaction between LRRC37B-FGF13A-SCN1B, LRRC37B-FGF13A, FGF13A-NAV1.6, SCN1B-NAV1.6, LRRC37B-SCN1B and/or the LRRC37B-FGF13A-SCN1B-NAV1.6 compared to a situation where the modulator is absent.
  • the modulator is an agonist of the LRRC37B-FGF13A-SCN1B, LRRC37B- FGF13A, FGF13A-NAV1.6, SCN1B-NAV1.6, LRRC37B-SCN1B and/or the LRRC37B-FGF13A-SCN1B-NAV1.6.
  • An enhanced interaction of LRRC37B and FGF13A or of FGF13A and NAVI.6, inhibits the NAVI.6 output and thus leads to a decreased neuron excitability.
  • An enhanced interaction of LRRC37B and SCN1B reduces the binding between SCN1B and NAVI.6 and thus lead to a decreased neuron excitability.
  • the modulator statistically significantly inhibits, decreases, reduces or blocks the interaction between LRRC37B-FGF13A, FGF13A-NAV1.6 and/or LRRC37B-SCN1B compared to a situation where the modulator is absent.
  • the modulator is an antagonist or inhibitor of the LRRC37B- FGF13A interaction, the FGF13A-NAV1.6 interaction or of the LRCC37B-SCN1B interaction.
  • Such reduced interaction overcomes FGF13A inhibition of NAVI.6 and thus lead to an increase neuron excitability, while a reduced interaction between LRRC37B and SCN1B leads to a reduced competition between SCN1B and NAVI.6 and thus to an increased neuron excitability.
  • said modulator of the interaction between the LRRC37B protein and the NAVI.6 voltage-gated sodium channel for use as a medicine is a protein or peptide, said protein or peptide comprising FGF13A or a FGF13A fragment or a peptidomimetic thereof.
  • said modulator is a peptide comprising a FGF13A fragment, said FGF13A fragment being an S- domain of FGF13A or FGF13A S-fragment or ExonS or a peptidomimetic thereof.
  • said FGF13A fragment comprises a peptide of SEQ. ID No.15 or SEQ ID No. 4 or a peptidomimetic thereof.
  • FGF13A fragment comprises a peptide of SEQ ID No.15.
  • said FGF13A fragment consists of SEQ ID No.15 or SEQ ID No. 4 or a peptidomimetic thereof.
  • the application provides a modulator of neuron excitability, wherein the modulator is the FGF13A protein or a peptidomimetic thereof, preferably a FGF13A protein fragment comprising or consisting of the S-fragment, referred herein to as a S-domain or ExonS, or a peptidomimetic thereof, the LRRC37B protein or a peptidomimetic thereof, the LRRC37B protein fragment comprising the B specific domain or a peptidomimetic thereof or any of the FGF13A, FGF13A S-fragments or LRRC37B proteins or protein fragments described herein.
  • the S-fragment and S-domain of the FGF13A protein or ExonS can be used as synonyms throughout the description.
  • FGF13A refers to the Fibroblast Growth Factor 13A (Uniprot ID: Q92913-1) and is also known as FHF2A (Fibroblast growth factor homologous factor). Its cDNA sequence is depicted in SEQ ID No. 1, while its amino acid sequence is depicted in SEQ ID No. 2.
  • the application provides the FGF13A protein as depicted in SEQ ID No. 2 or a homologue thereof.
  • a "homologue thereof" as used in current application refers to a protein or protein fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity compared to the reference protein (e.g. FGF13A protein as depicted in SEQ ID No. 2).
  • the homologue should comprise the same or similar effect as the reference protein (e.g. FGF13A protein as depicted in SEQ ID No. 2).
  • modifications to the amino acid sequences are still within the scope of the application as long as the homologue still has the same or similar effect (e.g. reducing NAVI.6 activity and therefore neuron excitability).
  • modifications comprise those known to the skilled person and include, for example, the substitution of one or more hydrophobic amino acids, preferably surface-exposed hydrophobic amino acids, with one or more hydrophilic amino acids.
  • the modifications comprise the substitution of up to 10, 9, 8, 7, 6, 5, 4, 3 or 2, hydrophobic amino acids, preferably surface-exposed hydrophobic amino acids, with hydrophilic amino acids.
  • a homologue consists of an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to amino acid sequence of the reference protein.
  • an FGF13A homologue is provided consisting of an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to SEQ ID No. 2.
  • the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.
  • the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.
  • Constant substitutions may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved.
  • the 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
  • conservative substitutions are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide.
  • glycine and proline may be substituted for one another based on their ability to disrupt a-helices.
  • non-conservative substitutions are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.
  • the substitutions may also include non-classical amino acids (e.g.
  • selenocysteine pyrrolysine, N-formylmethionine p-alanine, GABA and 6-Aminolevulinic acid
  • 4-aminobenzoic acid PABA
  • D-isomers of the common amino acids 2,4-diaminobutyric acid, a-amino isobutyric acid, 4- aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, p- alanine, fluoro-amino acids
  • nucleic acid sequence encoding the FGF13A protein as depicted in SEQ ID No. 2 or any of the homologues thereof described above is provided herein, more particularly the nucleic acid sequence as depicted in SEQ ID No. 1.
  • a host cell comprising the nucleic acid sequence, more particularly heterologously or recombinantly expressing the nucleic acid sequence.
  • a non-limiting example of a host cell is a HEK cell.
  • the application provides a FGF13A peptide or protein fragment of at most 100 amino acids, at most 90, at most 80 or at most 70 amino acids comprising the amino acid sequence as depicted in SEQ ID No. 4.
  • SEQ ID No. 4 depicts the 63 amino acids long sequence encoded by the S exon of the FGF13A gene, referred to herein as the FGF13A_S peptide or as FGF13A S-fragment or as the FGF13A S-domain or as ExonS.
  • the application provides the FGF13A protein fragment consisting of SEQ ID No. 4.
  • Said FGF13A protein fragment consisting of SEQ ID No. 4 is referred to as an FGF13A S-fragment or an ExonS in the present application.
  • a homologue is provided of the FGF13A S-fragment according to the definition above, hence comprising at least 90% amino acid identity with the FGF13A protein fragment as depicted in SEQ ID No. 4, wherein the homologue has the same or similar function or effect as the FGF13A protein S-fragment as depicted in SEQ ID No. 4.
  • an FGF13A protein fragment is provided consisting of an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to SEQ ID No. 4, wherein the FGF13A protein fragment still has the same or similar activity compared to SEQ ID No. 4.
  • the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.
  • the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.
  • a further fragment of FGF13A S-fragment (i.e. ExonS) is provided.
  • said fragment comprises a sequence as depicted in SEQ ID No. 15 or a homologue thereof.
  • homologue it is meant a peptide which differs in ten amino acids, six amino acids, five amino acids, four amino acids, three amino acids, preferably two amino acids, most preferably one amino acid to the SEQ ID No. 15.
  • a further fragment of said FGF13A S-fragment or ExonS is provided consisting of an amino acid sequence having one, or two, three, four, five, six or ten amino acid mutations with respect to SEQ ID No. 15.
  • the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.
  • the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.
  • Said fragment depicted as SEQ ID 15 consists of 24 amino acid long sequence, and is referred herein to as "ExonS 40-63".
  • a fragment of FGF13A S-fragment comprising SEQ ID No. 16 or a homologue thereof, preferably consisting of SEQ ID No. 16.
  • homologue it is meant a peptide which differs in ten amino acids, six amino acids, five amino acids, four amino acids, three amino acids, preferably two amino acids, most preferably one amino acid to the SEQ ID No. 16.
  • a further fragment of said FGF13A S-fragment or ExonS is provided consisting of an amino acid sequence having one, or two, three, four, five, six or ten amino acid mutations with respect to SEQ ID No. 16.
  • the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.
  • the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.
  • Said fragment depicted as SEQ ID 16 consists of 20 amino acid long sequence referred herein to as "ExonS 4-23".
  • SEQ ID No. 15 amino acid sequence FGF13A_S 40-63)
  • the amino acid sequence depicted in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 15 or SEQ ID No. 16 or the homologue thereof refers to an isolated protein or peptide.
  • the amino acid sequence depicted in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 15 or SEQ ID No. 16 or the homologue thereof is generated by chemical amino acid synthesis.
  • SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 15 or SEQ ID No. 16 or the homologue thereof is generated by recombinant production. It should be understood that a peptide of a sequence SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 15 or SEQ ID No. 16 could be provided by any suitable method known to a person skilled in the art, without departing from the scope of the present invention.
  • a molecule comprising the FGF13A protein as depicted in SEQ ID No. 2, or FGF13A fragment SEQ ID No. 4, SEQ ID No. 15 or SEQ ID No. 16, or a homologue thereof and an additional entity.
  • Said additional entity can be a biological, chemical or another protein or protein fragment.
  • said entity is a half-life extension entity and/or an entity that facilitates the molecule to cross the blood brain barrier.
  • said molecule is a chimeric molecule, a chimeric protein, a dimeric protein, a fusion protein, a composition, a combination, a peptide or a polypeptide.
  • compositions comprising an FGF13A protein fragment, wherein the FGF13A protein fragment is the FGF13A S-fragment as depicted in SEQ ID No. 4, or a FGF13A fragment comprising SEQ ID No.15 or SEQ ID No. 16 or any of the homologues thereof as described herein.
  • the pharmaceutical composition comprising the FGF13A fragment, said FGF13A fragment comprising SEQ ID NO. 15, preferably consisting of SEQ ID NO. 15 or a homologue thereof is provided.
  • a peptidomimetic of the FGF13A S-fragment or a peptidomimetic of a FGF13A fragment comprising SEQ ID No.15 or SEQ ID No. 16, is provided.
  • "Peptidomimetic" as used herein refers to a non-natural peptide or peptide comprising at least one non-natural amino acid. Peptidomimetics provide an alternative source of potent and selective Protein-Protein Interaction (PPI) modulators and occupy the chemical gap between small molecules and biologies, such as antibodies.
  • the FGF13A S-fragment or peptidomimetic thereof comprises at least one D-amino acid.
  • L- amino acids occur in all proteins produced by animals, plants, fungi and bacteria.
  • D-amino acids the amine group occurs in the right side in the Fisher projection.
  • D-Amino acids are only occasionally found in nature as residues in proteins.
  • the amino acids that make up the proteins in mammals are all L-amino acids. Hence, naturally occurring human proteins or peptides do not comprise D-amino acids.
  • Non-natural amino acids are so called because they are not found in natural polypeptide chains. They are not among the 20 amino acids attached to tRNAs in living cells used to polymerize proteins. Some unnatural amino acids do occur naturally, but most are chemically synthesized.
  • N-methyl amino acids attachment of a methyl group to the nitrogen in the amino group
  • a-methyl amino acids a methyl group replaces the hydrogen on the a carbon
  • beta-amino acids addition of a second carbon between the amino group and carboxy groups
  • homo-amino acids addition of a methylene group between the a carbon and the side group
  • beta-homo-amino acids addition of a second carbon between the amino and carboxy groups and the addition of a methylene group between the a carbon and the side group.
  • Unnatural amino acids are valuable building blocks in the manufacture of a wide range of pharmaceuticals.
  • Non-natural amino acids can exhibit biological activity as free acids and they can be incorporated into linear or cyclic peptides with biological activity.
  • the FGF13A S-fragment or the FGF13A fragment or peptidomimetic thereof comprises at least one non-natural amino acid.
  • Non-limiting examples of non-natural amino acids are isoethylmethyl-benzene, 6-chloro-3-methyl-lH-indole, methylcyclohexane, ethylcyclohexane, 2-naphthalene, ethylbenzene, l,l-difluoro-4-cyclohexyl, 4-methyl-l-methoxy-2- methylbenzene, l-chloro-4-methylbenzene, 4-methylphenyl-methanol, 3-methylbenzoic acid and 4- methylaniline.
  • the application also provides a nucleic acid molecule encoding the FGF13A S-fragment or FGF13A fragment or homologues thereof as described above, more particularly a nucleic acid molecule as depicted in SEQ. ID No. 3 or SEQ ID No. 17, respectively.
  • a vector is provided comprising the nucleic acid molecule encoding the FGF13A protein as depicted in SEQ ID No. 2 or the FGF13A fragments as depicted in SEQ ID No. 4 or SEQ ID No. 15.
  • a host cell comprising the nucleic acid molecule or the vector, more particularly heterologously expressing the nucleic acid molecule or with recombinant protein application.
  • a nonlimiting example of a host cell is a HEK cell.
  • the present application concerns an isolated FGF13A protein fragment, preferably an isolated FGF13A S fragment, more preferably a peptide comprising SEQ ID No. 15, a peptidomimetic thereof or a nucleic acid molecule encoding said FGF13A protein fragment, FGF13A S- fragment as depicted in SEQ ID No. 4 or the peptide comprising SEQ ID No. 15 for use as a medicine.
  • Said FGF13A protein fragment, FGF13A S-fragment and/or the peptide comprising SEQ ID No. 15 were found to be particularly efficient in modulating the neuronal excitability of human cortical pyramidal neurons.
  • FGF13A, the FGF13A protein fragment, the FGF13A S fragment and/or the peptide comprising SEQ ID No. 15 or SEQ ID No. 4 are used to decrease neuron excitability, even more particularly for use to treat seizures, epilepsy, epileptogenesis, ataxia, autism spectrum disorder, intellectual disability, cardiac arrythmia or pain.
  • LRRC37B acts at least in part through FGF13A on the NAVI.6 sodium channel.
  • FGF13A acts at least in part through FGF13A on the NAVI.6 sodium channel.
  • the interaction between FGF13A and LRRC37B is established through binding of the FGF13A S-fragment and LRR domain of LRRC37B respectively.
  • the LRR domain of LRRC37B is depicted in SEQ ID No. 8.
  • the application provides the LRRC37B protein as depicted in SEQ ID No. 6 or a homologue thereof, more particular a homologue of at least 90% identity over the full length of LRRC37B, wherein the homologue has the same or similar function as the LRRC37B domain as depicted in SEQ ID No. 6.
  • an LRRC37B protein is provided consisting of an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to SEQ ID No. 6, wherein the protein still has the same or similar activity compared to SEQ ID No. 6.
  • the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions and truncations.
  • the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.
  • a nucleic acid sequence is provided encoding the LRRC37B or homologue thereof, particularly encoding the amino acid sequence as depicted in SEQ ID No. 6, more particularly a nucleic acid sequence as depicted in SEQ. ID No. 5.
  • a host cell is provided comprising the nucleic acid sequence, more particularly heterologously expressing the nucleic acid sequence or with recombinant protein application.
  • a non-limiting example of a host cell is a HEK cell.
  • the application provides a LRRC37B protein fragment consisting of SEQ ID No. 8 or a homologue thereof, more particularly a homologue of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over the LRRC37B protein fragment's full length.
  • SEQ ID No. 8 depicts the amino acid sequence of the LRR domain of the LRRC37B protein, referred to herein as the LRRC37B-LRR peptide.
  • an LRRC37B protein fragment is provided consisting of an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to SEQ ID No. 8, wherein the LRRC37B protein fragment still has the same or similar activity compared to SEQ ID No. 8.
  • the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.
  • the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.
  • a molecule comprising the LRRC37B-LRR peptide as depicted in SEQ ID No. 8, or a homologue thereof and an additional entity.
  • Said additional entity can be a biological, chemical or another protein or protein fragment.
  • said entity is a half-life extension entity and/or an entity that facilitates the molecule to cross the blood brain barrier.
  • said molecule is a chimeric molecule, a chimeric protein, a dimeric protein, a fusion protein, a composition, a combination, a peptide or a polypeptide.
  • a peptidomimetic of the LRRC37B-LRR peptide comprising at least one non-natural amino acid and/or D-amino acid.
  • a pharmaceutical composition comprising the LRRC37B-LRR peptide as depicted in SEQ ID No. 8 or any of the homologues or peptidomimetics thereof as described herein.
  • the application also provides a nucleic acid molecule encoding the LRRC37B-LRR peptide or homologue thereof, particularly encoding the amino acid sequence as depicted in SEQ ID No. 8, more particularly a nucleic acid molecule as depicted in SEQ ID No. 7. Also provided is a vector comprising any of the nucleic acid molecules.
  • a host cell is also provided comprising the nucleic acid molecule or the vector, more particularly heterologously expressing the nucleic acid sequence or with recombinant protein application.
  • a non-limiting example of a host cell is a HEK cell.
  • SEQ ID No. 7 (cDNA of LRR domain from LRRC37B)
  • SEQ ID No. 8 amino acid sequence of LRR domain from LRRC37B
  • the present application concerns an isolated LRRC37B protein fragment, preferably an isolated LRRC37B fragment comprising SEQ ID No. 8 or SEQ ID No. 12, a peptidomimetic thereof or a nucleic acid molecule encoding said LRRC37B protein fragment, preferably LRRC37B comprising SEQ ID No. 8 or SEQ ID No. 12 for use as a medicine.
  • any of the FGF13A or LRRC37B proteins or protein fragments thereof or homologues thereof as described herein or nucleic acid molecules encoding them or vectors comprising the nucleic acid molecules are provided for use as a medicine, more particularly for use to modulate neuron excitability, even more particularly for use to decrease neuron excitability, even more particularly for use to treat seizures, epilepsy or epileptogenesis.
  • any of the peptidomimetics or fusion proteins or pharmaceutical compositions disclosed herein is provided for use as a medicine, more particularly for use to modulate neuron excitability, even more particularly for use to decrease neuron excitability, even more particularly for use to treat seizures, epilepsy or epileptogenesis.
  • the blood-brain barrier is a protective layer of tightly joined cells that lines the blood vessels of the brain which prevents entry of harmful substances (e.g. toxins, infectious agents) and restricts entry of (non-lipid) soluble molecules that are not recognized by specific transport carriers into the brain.
  • harmful substances e.g. toxins, infectious agents
  • drugs such as the FGF13A derived peptides or peptidomimetics or the LRRC37B derived peptides or peptidomimetics as described herein, to the central nervous system or brain in that drugs transported by the blood not necessarily will pass the blood-brain barrier.
  • drugs such as the FGF13A derived peptides or
  • Drugs can be directly injected into the brain (invasive strategy) or can be directed into the brain after BBB disruption with a pharmacological agent (pharmacologic strategy).
  • Invasive means of BBB disruption are associated with the risk of hemorrhage, infection or damage to diseased and normal brain tissue from the needle or catheter.
  • Direct drug deposition may be improved by the technique of convection- enhanced delivery.
  • Longer term delivery of a therapeutic protein can be achieved by implantation of genetically modified stem cells, by recombinant viral vectors, by means of osmotic pumps, or by means of incorporating the therapeutic drug in a polymer (slow release; can be implanted locally).
  • Pharmacologic BBB disruption has the drawback of being non-selective and can be associated with unwanted effects on blood pressure and the body's fluid balance. This is circumvented by targeted or selective administration of the pharmacologic BBB disrupting agent.
  • intra-arterial cerebral infusion of an antibody (bevacizumab) in a brain tumor was demonstrated after osmotic disruption of the BBB with mannitol (Boockvar et al. 2011, J Neurosurg 114:624-632); other agents capable of disrupting the BBB pharmacologically include bradykinin and leukotriene C4 (e.g. via intracarotid infusion; Nakano et al. 1996, Cancer Res 56:4027-4031).
  • BBB transcytosis and efflux inhibition are other strategies to increase brain uptake of drugs supplied via the blood.
  • Using transferrin or transferrin-receptor antibodies as carrier of a drug is one example of exploiting a natural BBB transcytosis process (Friden et al. 1996, J Pharmacol Exp Ther 278:1491-1498). Exploiting BBB transcytosis for drug delivery is also known as the molecular Trojan horse strategy.
  • Another mechanism underlying BBB, efflux pumps or ATP-binding cassette (ABC) transporters such as breast cancer resistance protein (BCRP/ABCG2) and P-glycoprotein (Pgp/MDRl/ABCBl)
  • BCRP/ABCG2 breast cancer resistance protein
  • Pgp/MDRl/ABCBl P-glycoprotein
  • Therapeutic drugs can alternatively be loaded in liposomes to enhance their crossing of the BBB, an approach also known as liposomal Trojan horse strategy.
  • a more recent and promising avenue for delivering therapeutic drugs to the brain consists of (transient) BBB disruption by means of ultrasound, more particularly focused ultrasound (FUS; Miller et al. 2017, Metabolism 69:S3-S7).
  • this technique has, often in combination with realtime imaging, the advantage of precise targeting to a diseased area of the brain.
  • Therapeutic drugs can be delivered in e.g. microbubbles e.g. stabilized by an albumin or other protein, a lipid, or a polymer.
  • Therapeutic drugs can alternatively, or in conjunction with microbubbles, be delivered by any other method, and subsequently FUS can enhance local uptake of any compound present in the blood (e.g. Nance et al.
  • Microbubbles with a therapeutic drug load can also be induced to burst (hyperthermic effect) in the vicinity of the target cells by means of FUS, and when driven by e.g. a heat shock protein gene promoter, localized temporary expression of a therapeutic protein can be induced by ultrasound hyperthermia (e.g. Lee Titsworth et al. 2014, Anticancer Res 34:565-574).
  • ultrasound hyperthermia e.g. Lee Titsworth et al. 2014, Anticancer Res 34:565-574
  • Alternatives for ultrasound to induce the hyperthermia effect are microwaves, laser-induced interstitial thermotherapy, and magnetic nanoparticles (e.g. Lee Titsworth et al. 2014, Anticancer Res 34:565-574).
  • screening methods are provided to identify modulators of the LRRC37B-FGF13A- NAV1.6 complex or alternatively phrased to identify modulators of neuronal excitability.
  • the modulator increases or reduces the LRRC37B-FGF13A interaction compared to a situation in the absence of the modulator.
  • the application thus provides a method, more particularly an in vitro method, of identifying a modulator of neuronal excitability or a modulator of the LRRC37B- FGF13A interaction, said method comprises the steps of: i) providing the LRRC37B protein, or a fragment thereof comprising or consisting of the LRR domain as depicted in SEQ ID No. 8; ii) providing the FGF13A protein or a fragment thereof comprising or consisting of the fragment as depicted in SEQ ID No. 15 or SEQ ID No.
  • iii) providing a test compound that is candidate for being a modulator of neuronal excitability or of the LRRC37B-FGF13A interaction; iv) contacting LRRC37B or the fragment thereof provided in i) with the FGF13A protein or the fragment thereof provided in ii) in the presence or absence of the test compound provided in iii); v) identifying as modulator of neuronal excitability or of the LRRC37B-FGF13A interaction, from iv) the test compound that is, compared to identical conditions but in absence of said test compound, statistically significantly reducing or increasing the binding of the FGF13A protein or the fragment thereof to LRRC37B or the fragment thereof.
  • test compound can be considered as modulator of the LRRC37B-FGF13A interaction if said test compound can statistically significantly reduce or increase the binding of the FGF13A protein or the fragment thereof to LRRC37B or the fragment thereof, compared to the identical conditions in absence of said test compound.
  • the test compound that reduces the binding between FGF13A and LRRC37B protein or fragments thereof shows a higher affinity to LRRC37B than FGF13A and therefore competes with FGF13A for binding to LRRC37B.
  • the test compound is a peptide consisting of 40, 30, 20, 15 or 10 contiguous amino acids or less, wherein the peptide is a fragment of FGF13A-S fragment as depicted SEQ ID No. 4, preferably the fragment as depicted in SEQ ID No. 15 or the test compound is a peptidomimetic of said peptide.
  • the binding of LRRC37B or the fragment thereof to the FGF13A protein or the fragment thereof is determined by immunologic, fluorescent or radiologic detection, co-sedimentation, co-immunoprecipitation, or electron microscopy.
  • Assays for studying receptor binding are well-known by the person skilled in the art.
  • a non-limiting example is the fluorescence polarization (FP) assay.
  • FP fluorescence polarization
  • the assay is based on the rotational movement of fluorescently labelled molecules in solution. Unbound molecules rotate rapidly, are therefore randomly orientated prior to light emission and hence show a low polarization value. However, if the rotation of a fluorescently labelled molecule is slowed down because it binds to a large complex, it shows a high polarization value.
  • the LRRC37B or the fragment thereof in step i) is provided by one or more cells expressing LRRC37B or the fragment thereof.
  • the FGF13A or the fragment thereof in step ii) is administered extracellularly to the one or more cells expressing LRRC37B or the fragment thereof.
  • the present invention relates to an in vitro method of identifying an LRRC37B binding peptide, said method comprises the steps of: i) Providing a LRRC37B protein, or a fragment thereof comprising or consisting of a LRR domain comprising or consisting of SEQ ID No. 8; ii) providing a FGF13A protein fragment derived from a FGF13A S-fragment, preferably the FGF13A fragment comprising or consisting of SEQ ID No. 15 or SEQ ID No. 4 or generating a peptidomimetic derived from a FGF13A S-fragment, preferably the FGF13A fragment comprising or consisting of SEQ ID No. 15 or SEQ ID No.
  • a method is provided of identifying a modulator of neuronal excitability or a LRRC37B binding peptide, said method comprises the steps of: i) providing the LRRC37B protein, or a fragment thereof comprising or consisting of the LRR domain as depicted in SEQ ID No. 8; ii) providing a plurality of FGF13A protein fragments derived from the FGF13A S-fragment as depicted in SEQ ID No. 4 or SEQ ID No. 15 or generating a plurality of peptidomimetics derived from the FGF13A S-fragment as depicted in SEQ ID No. 4 or SEQ ID No.
  • the binding of the LRRC37B protein or the fragment thereof to the FGF13A protein or the fragment thereof is determined by immunologic or radiologic detection, co-sedimentation, coimmunoprecipitation or electron microscopy.
  • said binding may be determined by any suitable method known in the prior art without departing from the scope of the present invention.
  • the LRRC37B protein or the fragment thereof can be provided by cells expressing the LRRC37B protein or the fragment thereof, preferably LRRC37B protein or the fragment thereof is provided by cells expressing the LRRC37B protein or the fragment thereof.
  • the FGF13A protein or the fragment or peptidomimetic thereof can be administered extracellularly to the cells expressing the LRRC37B protein or the fragment thereof, preferably the FGF13A protein or the fragment or peptidomimetic thereof is administered extracellularly to the cells expressing the LRRC37B protein or the fragment thereof.
  • NAVI.6 (UniProt ID: Q9UQD0; also termed SCN8A or PN4) is a voltage-gated sodium channel a subunit that is the most abundantly expressed isoform in the CNS during adulthood and is enriched at the axon initial segment and at the nodes of Ranvier. The channels are highly concentrated in sensory and motor axons in the peripheral nervous system. NAVI.6 facilitates action potential propagation when the membrane potential is depolarized by an influx of Na+ ions. However, NAVI.6 is able to sustain repetitive excitation and firing.
  • NAVI.6 The high frequency firing characteristic of NAVI.6 is caused by a persistent and resurgent sodium current. This characteristic is caused by slow activation of the sodium channel following repolarization, which allows a steady-state sodium current after the initial action potential propagation.
  • NAVI.6 is tetradotoxin (TTX)-sensitive.
  • TTX tetradotoxin
  • SEQ ID No. 14 amino acid sequence of NAVI.6
  • the modulator of neuronal excitability as provided herein increases or reduces the FGF13A-NAV1.6 interaction.
  • the application thus provides a method, more particularly an in vitro method, of identifying a modulator of neuronal excitability or a modulator of the
  • said method comprises the steps of: i) providing the FGF13A protein, or a fragment thereof comprising or consisting of the S protein fragment as depicted in SEQ ID No. 4, preferably as depicted in SEQ ID No. 15, or a peptidomimetic thereof; ii) providing the NAVI.6 protein as depicted in SEQ ID No.
  • immune-based assays are comprised in the claimed method, more specifically "immune-based assays” or “immune-based detection” for monitoring the binding between protein fragments or between compounds and one or more proteins.
  • immunosorbent-based assays comprise the most broadly used bio-detection technologies that are based on the use of antibodies, and are well known in the art. Antibodies are highly suited for detecting small quantities of target proteins in the presence of complex mixtures of proteins.
  • an “immune-based assay”, “immunoassay” or “immune-based detection” refers to a biochemical binding assay involving binding between antibodies and antigen, which measures the presence or concentration of a substance in a sample, such as a biological sample, using the reaction of an antibody to its cognate antigen, for example the specific binding of an antibody to a protein. Both the presence of the antigen or the amount of the antigen present can be measured.
  • immunoassays are enzyme linked immunosorbent assays (ELISAs), enzyme linked immunospot assay (ELISPOT), immunobead capture assays, Western blotting, gel-shift assays, protein arrays, multiplexed bead arrays, magnetic capture, fluorescence resonance energy transfer (FRET), a sandwich assay, a competitive assay, an immunoassay using a biosensor, an immunoprecipitation assay etc.
  • ELISAs enzyme linked immunosorbent assays
  • ELISPOT enzyme linked immunospot assay
  • immunobead capture assays Western blotting, gel-shift assays, protein arrays, multiplexed bead arrays, magnetic capture, fluorescence resonance energy transfer (FRET), a sandwich assay, a competitive assay, an immunoassay using a biosensor, an immunoprecipitation assay etc.
  • FRET fluorescence resonance energy transfer
  • the present screening methods might be based on a combination or a series of measurements, particularly when establishing the link between the impairment of the activity of the NAVI.6 channel by specific test compounds. Also, it should be clear that there is no specific order in performing these measurements while practicing the present invention.
  • immune-based assays involve contacting a sample suspected of containing a molecule of interest (such as the test compound) with an antibody to the molecule of interest or contacting an antibody to a molecule of interest with a molecule that can be bound by the antibody, as the case may be, under conditions effective to allow the formation of immune complexes.
  • a sample suspected of containing a molecule of interest such as the test compound
  • an antibody to the molecule of interest or contacting an antibody to a molecule of interest with a molecule that can be bound by the antibody, as the case may be, under conditions effective to allow the formation of immune complexes.
  • the sample-antibody composition such as a tissue section, ELISA plate, dot blot or Western blot, can then be washed to remove any non- specif ically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
  • Immunoassays can include methods for detecting or quantifying the amount of a molecule of interest in a sample, which methods generally involve the detection or quantitation of any immune complexes formed during the binding process.
  • the immune-based detection is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or any other known label. See, for example, U.S. Pat. Nos.
  • a label can include a fluorescent dye, a member of a binding pair, such as biotin/streptavidin, a metal (e.g., gold), or an epitope tag that can specifically interact with a molecule that can be detected, such as by producing a coloured substrate or fluorescence.
  • Substances suitable for detectably labelling proteins include fluorescent dyes (also known herein as fluorochromes and fluorophores) and enzymes that react with colorimetric substrates (e.g., horseradish peroxidase).
  • fluorescent dyes also known herein as fluorochromes and fluorophores
  • enzymes that react with colorimetric substrates e.g., horseradish peroxidase.
  • the use of fluorescent dyes is generally preferred in the practice of the invention as they can be detected at very low amounts.
  • each antigen can be labelled with a distinct fluorescent compound for simultaneous detection. Labelled spots on the array are detected using a fluorimeter, the presence of a signal indicating an antigen bound to a specific antibody.
  • Fluorophores are compounds or molecules that luminesce. Typically, fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength.
  • ELISA is a heterogeneous immunoassay, which can be used in the methods disclosed herein.
  • the assay can be used to detect protein antigens in various formats. In the "sandwich" format the antigen being assayed is held between two different antibodies. In this method, a solid surface is first coated with a solid phase antibody.
  • the in vitro system composition comprises the antigen, to which the test compound is added, allows binding of the test compound, and therefore reduces the detection of the antigen via reaction with the bound antibody. Any unbound antigen is washed away. A known amount of enzyme-labelled antibody is then allowed to react with the bound antigen.
  • ELISA can also be used as a competitive assay.
  • the test specimen containing the antigen to be determined is mixed with a precise amount of enzyme-labelled antigen and both compete for binding to an anti-antigen antibody attached to a solid surface. Excess free enzyme-labelled antigen is washed off before the substrate for the enzyme is added.
  • the amount of colour intensity resulting from the enzymesubstrate interaction is a measure of the amount of antigen in the sample tested.
  • a heterogeneous immunoassay such as an ELISA, can be used to detect any of the proteins disclosed or incorporated by reference herein. In many immunoassays, as described elsewhere herein, detection of antigen is made with the use of antigens specific antibodies as detector molecules. However, immunoassays and the systems and methods of the present invention are not limited to the use of antibodies as detector molecules. Any substance that can bind or capture the antigen within a given sample may be used. Aside from antibodies, suitable substances that can also be used as detector molecules include but are not limited to enzymes, peptides, proteins, and nucleic acids.
  • the captured antigen may be detected.
  • enzyme-linked antibodies produce a colour change.
  • detection of the captured antigen is made through detecting fluorescent, luminescent, chemiluminescent, or radioactive signals.
  • the system and methods of the current invention is not limited to the particular types of detectable signals produced in an immunoassay.
  • a method of identifying a modulator of neuron excitability comprising the steps of: generating a peptide consisting of less than 30 contiguous amino acids wherein the peptide is a fragment of FGF13A S-fragment as depicted in SEQ ID No. 4, preferably said peptide comprising SEQ ID No. 15, or generating a peptidomimetic of said peptide; providing a NAVI.6 protein as depicted in SEQ ID No.
  • a NAVI.6 protein or the fragment thereof contacting a NAVI.6 protein or the fragment thereof with said peptide or peptidomimetic; identifying as a modulator of neuron excitability a peptide or peptidomimetic that, compared to identical conditions but in absence of the peptide or peptidomimetic, statistically significantly reduces or increases the activity of the NAVI.6 protein.
  • said NAVI.6 protein or the functional fragment thereof is provided by cells expressing NAVI.6 or the functional fragment thereof.
  • said cells are electrically excitable cells.
  • a method of identifying a modulator of neuron excitability comprising the steps of: i) generating a plurality of FGF13A protein fragments comprising the FGF13A fragment as depicted in SEQ ID No. 15 or SEQ ID No. 4 or derived from the FGF13A fragment as depicted in SEQ ID No. 15 or SEQ ID No. 4 or generating a plurality of peptidomimetics derived from the FGF13A fragment as depicted in SEQ ID No. 15 or SEQ ID No. 4; ii) providing the NAVI.6 protein as depicted in SEQ ID No.
  • iii) contacting the NAVI.6 protein provided in ii) with one or more of the FGF13A protein fragment or peptidomimetics provided in step i); iv) identifying as a modulator of neuron excitability from iii) a FGF13A protein fragment or peptidomimetic that, compared to identical conditions but in absence of the FGF13A protein fragment or peptidomimetic, statistically significantly reduces or increases the activity of the NAVI.6.
  • the FGF13A protein fragment is a part of the FGF13A S- fragment and is thus shorter in length than the FGF13A S-fragment.
  • said FGF13A protein fragment derived from the FGF13A S-fragment consists of 60, 50, 40, 30, 20, 10 or less contiguous amino acids.
  • the FGF13A protein fragments or peptidomimetics generated in step i) consist of at most 4, 5, 6, 7, 8, 9, 10, 15, 20 or at most 25 amino acids.
  • Also provided is a method, more particularly an in vitro method, of identifying a modulator of neuronal excitability comprising the steps of: - providing one or more excitable cells expressing the NAVI.6 protein as depicted in SEQ ID No.
  • peptide is a fragment of the FGF13A S-fragment as depicted in SEQ ID No. 4 or SEQ ID No. 15 or adding a peptidomimetic of said peptide to the one or more cells;
  • the compound statistically significantly reduces or increases the activity of NAVI.6 or the fragment thereof.
  • said FGF13A protein fragment, peptide or peptidomimetic shows a higher affinity to NAVI.6 than FGF13A and therefore competes with FGF13A for binding to and thus modulation of NAV1.6.
  • the NAVI.6 or the functional fragment thereof (e.g. in step ii)) is provided by cells expressing NAVI.6 or the functional fragment thereof. More particularly, said cells are excitable cells, more particular electrically excitable cells, even more particularly neuron or cardiac cells. In a further embodiment, identifying the modulator of NAVI.6 activity is based on an increased or decreased electrical excitability of the cells.
  • assays for determining activity of ion channels are straightforward and there are several fluorescent and non-fluorescent methods available (Birch et al 2004 DDT 9) that are amenable to high- throughput screening in 96-and 384-well plate arrays, as well as higher well densities.
  • Non-fluorescent methods for example directly measure the flux of an ion through the channel of interest and in some cases exploit the non-selective conductance of ions by the channel under investigation.
  • the ion flux can either be measured using radiotracers (e.g. 22Na or [14C]-guanidinium for sodium channels) or atomic absorption spectroscopy (AAS) for the detection of non-radioactive metal ions.
  • radiotracers e.g. 22Na or [14C]-guanidinium for sodium channels
  • AAS atomic absorption spectroscopy
  • the above screening method is provided wherein the NAVI.6 voltage-gated sodium channel is provided in a structure that separates a first medium from a second medium, wherein NAVI.6 exhibits independent ion permeation.
  • the amount of the independent ion permeation through the NAVI.6 voltage-gated sodium channel is then measured between the first and second media and compared between presence and absence of a potential modulator of NAVI.6 activity.
  • a method for identifying a modulator of the NAVI.6 voltage-gated sodium channel or of neuron excitability comprising the following steps: providing the NAVI.6 voltage-gated sodium channel in a structure that separates a first medium from a second medium, wherein NAVI.6 exhibits independent ion permeation; contacting the NAVI.6 voltage-gated sodium channel with a FGF13A derived peptide or peptidomimetic; measuring the amount of the independent ion permeation through the NAVI.6 voltage-gated sodium channel between the first and second media; and comparing the amount of the independent ion permeation measured for the NAVI.6 voltagegated sodium channel contacted with the FGF13A derived peptide or peptidomimetic to the amount of said independent ion permeation measured for the NAVI.6 voltage-gated sodium channel not contacted with the FGF13A derived peptide or peptidomimetic, wherein an increase or decrease in the amount of
  • the NAVI.6 activity refers to the ability of NAVI.6 to mediate a sodium current across a membrane.
  • the FGF13A derived peptide or peptidomimetic is a peptide of at most 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or at most 60 amino acids and consisting of a fragment of the FGF13A S-domain as depicted in SEQ ID No. 4 or a fragment as depicted in SEQ ID No. 15.
  • the FGF13A derived peptide or peptidomimetic is a peptide consisting of 20, 15 or 10 contiguous amino acids or less, wherein the peptide is a fragment of the FGF13A S-fragment as depicted in SEQ ID No. 15 or SEQ ID No. 4.
  • measuring the amount of independent sodium ion permeation through the voltage-gated sodium channel between the first and second media is by patch-clamp measurement. In other embodiments, measuring the amount of independent sodium ion permeation through the voltagegated sodium channel between the first and second media is by fluorescence measurement. In certain embodiments, measuring the amount of independent sodium ion permeation through the voltage-gated sodium channel between the first and second media is by radiolabeled measurement. In certain embodiments, measuring the amount of independent sodium ion permeation through the voltage-gated sodium channel between the first and second media is by biological assay measurement. Assays can be performed in an in vitro system. Therefore, in one embodiment, said cell expressing a functional NAVI.6 is an in vitro system comprising neuronal cells expressing a functional NAVI.6 channel.
  • said cell expressing a functional NAVI.6 channel is selected from a recombinant cell, a neuronal cell or a primary neuron.
  • said cell expressing a functional NAVI.6 channel is a neuron present in an acute brain slice derived from a non-human mammal.
  • Permeation refers to the rapid and selective transport of (sodium) ions through cell membranes (e.g. through sodium channels) which is essential for initiating action potentials within excitable cells.
  • excitable cell refers to a cell or cells with the ability to be electrically excited resulting in the generation of action potentials.
  • excitable cells are neurons, muscle cells (skeletal, cardiac, and smooth), and some endocrine cells (e.g. insulin-releasing pancreatic p cells).
  • the membrane potential of an excitable cell is polarized and can, in a non-limiting embodiment, be in the range of about -60mV to about -80mV.
  • Current application also provides methods for designing, selecting, screening and/or optimizing a chemical entity that binds to and/or modulates the activity of the NAVI.6 voltage-gated sodium channel, said methods comprise the steps of: a. generating a co-crystal comprising the NAVI.6 protein as depicted in SEQ ID No. 14 or fragment thereof and the FGF13A fragment as depicted in SEQ ID No. 15 or SEQ ID No. 4 or generating a co-crystal comprising the LRRC37B fragment as depicted in SEQ ID No. 8 and the FGF13A fragment as depicted in SEQ ID No. 15 or SEQ ID No. 4; b.
  • determining the three-dimensional atomic coordinates of the co-crystal by X-ray diffraction c. providing the three-dimensional atomic coordinates of the co-crystal on a computer comprising the means for generating three-dimensional structural information from atomic coordinates; and b. designing, selecting, screening and/or optimizing the chemical entity by performing a fitting operation between the chemical entity and the three-dimensional structural information of all or part of said crystal.
  • binding operation refers to an operation that utilizes the atomic coordinates of a chemical entity, binding pocket, molecule or molecular complex, or portion thereof, to associate the chemical entity with the binding pocket, molecule or molecular complex, or portion thereof. This may be achieved by positioning, rotating or translating a chemical entity in the binding pocket to match the shape and electrostatic complementarity of the binding pocket. Covalent interactions, non-covalent interactions such as hydrogen bond, electrostatic, hydrophobic, van der Waals interactions, and non-complementary electrostatic interactions such as repulsive charge-charge, dipole-dipole and charge-dipole interactions may be optimized. Alternatively, one may minimize the deformation energy of binding of the chemical entity to the binding pocket.
  • the present invention provides a modulator of the interaction between LRRC37B and SCN1B for use as a medicine.
  • Said modulator can be particularly advantageous for use in modulating, i.e. decreasing or increasing the neuronal excitability of human neurons, preferably human cortical pyramidal neurons.
  • said modulator can be used to decrease neuron excitability, even more particularly for use to treat seizures, epilepsy, epileptogenesis, ataxia, autism spectrum disorder, intellectual disability, cardiac arrythmia or pain.
  • LRRC37B binds SCN1B, a NAV p-subunit that modulates VGSCs, more particularly NAVI.6 (Wimmer et al 2010 J Clin Invest 120: 2661-2671). Interestingly, it was found that the LRRC37B competes with NAVI.6 for binding with SCN1B. As such expressing LRRC37B reduces or inhibits the binding between SCN1B and NAVI.6 and therefore decreases neuronal excitability of cortical neurons.
  • the application provides a modulator of neuron excitability or more particularly a modulator of the NAV1.6-SCN1B interaction, wherein the modulator is a LRRC37B protein fragment comprising the B specific domain as depicted in SEQ ID No. 12 or a homologue thereof.
  • SCN1B The nucleic acid sequence encoding SCN1B is depicted in SEQ ID No. 9 and the amino acid sequence is depicted in SEQ ID No. 10.
  • SEQ ID No. 11 depicts the nucleic acid sequence encoding the B specific domain of LRRC37B
  • SEQ ID No. 12 depicts the amino acid sequence of the B specific domain of LRRC37B.
  • an isolated LRRC37B protein fragment comprising or consisting of the amino acid sequence as depicted in SEQ ID No. 12 or a peptidomimetic generated from said fragment is provided.
  • a nucleic acid molecule encoding the isolated LRRC37B protein fragment is provided.
  • a host cell is also provided comprising the nucleic acid sequence, more particularly, heterologously expressing the nucleic acid sequence or with recombinant protein application.
  • a non-limiting example of a host cell is a HEK cell.
  • SEQ ID No. 10 amino acid sequence SCN1B
  • SEQ ID No. 11 (cDNA from LRRC37B specific domain)
  • SEQ ID No. 12 amino acid sequence from LRRC37B specific domain
  • the application provides an LRRC37B peptide or protein fragment of at most 100 amino acids, at most 90, at most 80, at most 70 or at most 60 amino acids comprising the amino acid sequence as depicted in SEQ ID No. 12.
  • SEQ ID No. 12 depicts a 54 amino acids long sequence comprising an LRRC37B specific domain, referred to herein as the "LRRC37B specific fragment” or "B specific fragment” or "LRRC37B specific domain” or "B specific domain”.
  • the application provides an LRRC37B protein fragment consisting of SEQ ID No. 12.
  • LRRC37B specific fragment comprising at least 90%, 91%, 92%; 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the LRRC37B specific fragment as depicted in SEQ ID No. 12, wherein the homologue has the same or similar function as the LRRC37B specific fragment as depicted in SEQ ID No. 12.
  • an LRRC37B protein fragment is provided consisting of an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to SEQ ID No.
  • the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.
  • the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.
  • the amino acid sequence depicted in SEQ ID No. 12 or the homologue thereof refers to an isolated protein or peptide. According to another embodiment, the amino acid sequence depicted in SEQ ID No. 12 or the homologue thereof is generated by chemical amino acid synthesis. According to another embodiment SEQ ID No. 12 or the homologue thereof is generated by recombinant production.
  • a molecule comprising the LRRC37B specific fragment as depicted in SEQ ID No. 12 respectively, or a homologue thereof and an additional entity.
  • Said entity can be a biological, chemical or another protein or protein fragment.
  • said entity is a half-life extension entity and/or an entity that facilitates the molecule to cross the blood brain barrier.
  • said molecule is a chimeric molecule, a chimeric protein, a dimeric protein, a fusion protein, a composition, a combination, a peptide or a polypeptide.
  • a fusion protein or a pharmaceutical composition comprising an LRRC37B protein fragment, wherein the LRRC37B protein fragment is the LRRC37B specific fragment as depicted in SEQ ID No. 12 or any of the homologues thereof as described herein.
  • a peptidomimetic of the LRRC37B specific fragment is provided.
  • the peptidomimetic comprises at least one D-amino acid and/or at least one non-natural amino acid.
  • composition comprising any of the above LRRC37B protein fragments or homologue or peptidomimetics thereof.
  • the application also provides a nucleic acid molecule encoding the LRRC37B specific fragment or homologue thereof as described above, more particularly a nucleic acid molecule as depicted in SEQ ID No. 11.
  • a vector is provided comprising the nucleic acid molecule encoding the LRRC37B specific fragment as depicted in SEQ ID No. 12 or as depicted in SEQ ID No. 11.
  • a host cell comprising the nucleic acid molecule or vector, more particularly heterologously expressing the nucleic acid sequence or with recombinant protein application.
  • a nonlimiting example of a host cell is a HEK cell.
  • any of the LRRC37B protein fragments comprising the B specific domain or homologues thereof as described herein or nucleic acid molecules encoding them or vectors comprising the nucleic acid molecules are provided for use as a medicine, more particularly for use to modulate neuron excitability, even more particularly for use to decrease neuron excitability, even more particularly for use to treat seizures, epilepsy or epileptogenesis.
  • any of the peptidomimetics or fusion proteins or pharmaceutical compositions disclosed herein is provided for use as a medicine, more particularly for use to modulate neuron excitability, even more particularly for use to decrease neuron excitability, even more particularly for use to treat seizures, epilepsy or epileptogenesis.
  • screening methods are provided to identify modulators of the LRRC37B-SCN1B interaction or alternatively phrased to identify modulators of neuronal excitability.
  • the modulator increases or reduces the LRRC37B-SCN1B interaction.
  • the application thus provides a method, more particularly an in vitro method, of identifying a modulator of neuronal excitability or a modulator of the LRRC37B-SCN1B interaction, said method comprises the steps of: i) providing LRRC37B, or a fragment thereof comprising or consisting of the B-specific domain as depicted in SEQ ID No. 12; ii) providing SCN1B protein as depicted in SEQ ID No.
  • test compound that is candidate for being a modulator of neuronal excitability or of the LRRC37B-SCN1B interaction or providing a peptidomimetic derived from the B-specific domain as depicted in SEQ ID No.
  • the binding of LRRC37B or the fragment thereof to the SCN1B protein or the fragment thereof is determined by immunologic, fluorescent or radiologic detection, co-sedimentation, co-immunoprecipitation, or electron microscopy.
  • any other method to determine the binding known in the prior art can be used.
  • Assays for studying protein-protein interactions are well- known by the person skilled in the art. A non-limiting example is the fluorescence polarization (FP) assay.
  • the LRRC37B or the fragment thereof in step i) is provided by one or more cells expressing LRRC37B or the fragment thereof.
  • a method of identifying a modulator of neuron excitability comprising the steps of: generating a peptide consisting of less than 30 contiguous amino acids wherein the peptide is a fragment of a LRRC37 B-specific domain as depicted in SEQ ID No. 12 or generating a peptidomimetic of said peptide; providing a SCN1B protein as depicted in SEQ ID No. 10 or a functional fragment thereof; contacting the SCN1B protein with the peptide or peptidomimetic or with the LRRC37 B- specific domain as depicted in SEQ ID No.
  • identical conditions it is meant that the binding of the SCN1B protein with the peptide or peptidomimetic is done under the same conditions, and using the same parameters, such as concentration, assay, temperature, etc. as the conditions in which the binding of the LRRC37 B-specific domain as depicted in SEQ ID No. 12 to SCN1B compared was determined.
  • a method of identifying a modulator of neuron excitability comprising the steps of: i) generating a plurality of peptides comprising 30, 25, 20, 15 or 10 or less contiguous amino acids wherein the peptides are fragments of the LRRC37 B-specific domain as depicted in SEQ ID No. 12 or generating a plurality of peptidomimetics from said peptides; ii) providing the SCN1B protein as depicted in SEQ ID No.
  • iii) contacting the SCN1B protein provided in ii) with one or more of the peptides or peptidomimetics provided in step i); iv) identifying as a modulator of neuron excitability from iii) a peptide or peptidomimetic that, compared to identical conditions shows a statistically significantly increased binding to SCN1B compared to the LRRC37 B-specific domain.
  • the method comprises an additional step of providing the NAVI.6 protein as depicted in SEQ ID No. 14 or a fragment thereof that binds SCN1B, wherein the NAVI.6 protein or fragment thereof is contacted with the SCN1B and wherein one or more of the LRRC37B peptides or peptidomimetics are added to the protein complex.
  • the one or more peptides or peptidomimetics are then identified as a modulator of neuron excitability when the peptide or peptidomimetic statistically significantly reduces the binding between SCN1B and the NAVI.6 protein or fragment thereof.
  • Current application also provides methods for designing, selecting, screening and/or optimizing a chemical entity that binds to and/or modulate the activity of the NAVI.6 channel, said methods comprise the steps of: a. generating a co-crystal comprising the SCN1B protein and the LRRC37B specific fragment as depicted in SEQ ID No. 12 or a co-crystal comprising the SCN1B protein and the NAVI.6 protein as depicted in SEQ ID No. 14 or fragment thereof that binds SCN1B; b. determining the three-dimensional atomic coordinates of the co-crystal by X-ray diffraction; c.
  • Example 1 LRRC37 gene family is selectively expressed in human cortical neurons
  • the LRRC37 (Leucine-Rich Repeat-containing protein 37) gene family encodes putative orphan receptors of unknown function.
  • the number of LRRC37 genes increased in simian species leading to 3 encoding paralogs in the chimpanzee and 4 in the human genomes, compared to two in the mouse genome.
  • LRRC37 genes encode A and B types of transmembrane proteins comprising an LRR in their extracellular part.
  • the B type LRRC37 carries a specific domain, hereafter referred to as B domain or the LRRC37B specific domain, located near the N-terminal part of the protein.
  • the LRRC37B type emerged in the simian genomes.
  • LRRC37B at the protein level (uniprot ID: Q96QE4) in the adult human cerebral cortex, using a specific antibody that only recognizes the B and not the A types. Remarkably this revealed a selective localization of LRRC37B at the level of the axon initial segment (AIS - as determined with AIS marker Ankyrin G) of a fraction of pyramidal neurons (Figure 2A-B). Time-course analysis at several stages from neonatal to >60 years old specimens, revealed that the proportion of neurons sharply increased from birth until childhood to stabilize at puberty (Figure 2F).
  • LRRC37B is a human cortical neuron-specific protein located at the axon initial segment.
  • Example 2 LRRC37B negatively regulates cortical pyramidal neuron excitability
  • LRRC37B In order to probe the function of LRRC37B, we first performed gain-of-function in mouse pyramidal neurons through sparse in utero electroporation in the cortex (at embryonic day 15.5, thereby targeting mostly cortical layer 2-3 pyramidal neurons), followed by analysis at P21. We then detected the LRRC37B protein enriched at the axon initial segment (AIS), colocalized with ankyrin-G ( Figure 2C). We detected no differences in the AIS length and location compared with control neurons.
  • AIS axon initial segment
  • Figure 2C We detected no differences in the AIS length and location compared with control neurons.
  • the AIS is a subcellular compartment critical for neuronal excitability, as the main site of generation of action potentials (AP) through the opening of sodium-voltage gated channel a subunits (NAVa) concentrated at this site (Leterrier 2016 Curr Top Membr 77, 185-233).
  • AP action potential
  • NAVa subunits
  • LRRC37B gain-of-function led to a striking decrease in neuronal excitability, characterized by a lower AP firing rate in current clamp (Figure 2D(a)), a higher rheobase and an increased AP risetime and AP width (Figure 2E).
  • the AIS is specifically targeted by chandelier GABA-ergic interneurons that thereby regulate pyramidal cell output (Gallo et al 2020 Trends Neurosci 43, 565-580).
  • patch-clamp recordings detected no differences in excitatory and inhibitory postsynaptic potentials and quantification of GABA-ergic synapses at the level of AIS detected no differences either.
  • LRRC37B acts as a negative regulator of neuronal excitability, mostly at an intrinsic level.
  • Example 3 LRRC37B binds to FGF13A ligand, and thereby negatively regulates voltage gated channels
  • FGF13 was previously described as a member of the FHF non-canonical FGF family, which is thought to encode non-secreted proteins (Dover et al 2010 J Physiol 588, 3695-3711; Smallwood et al 1996 Proc Natl Acad Sci USA 93, 9850-9857; Wang et al 2012 Structure 20, 1167-1176).
  • FGF13 encodes several splicing isoforms (FGF13A, FGF13B, FGF13V, FGF13Y and FGF13VY) that share C terminal exons (regions 2-5 as visualized in Figure 3A upper panel, "core domain") and differ by their initial first exon (Munoz-Sanjuan et al 2000 J Biol Chem 275, 2589-2597) (Figure 3A).
  • FGF13A splicing isoforms
  • FGF13A is an extracellular ligand of LRRC37B, to which it binds to through its N-terminal domain.
  • LRRC37B thus constitutes a selective receptor for FGF13A, to which it binds to through its LRR domain.
  • Example 3(b) Determining the minimal binding fragment of FGF13A S-domain (minimal binding fragment of "Exon S")
  • FGF13A More particularly FGF13A S-domain or S-fragment or ExonS
  • LRRC37B receptor responsible for the interaction with FGF13A
  • affinity measures using fluorescent polarization assay between recombinant protein LRR and synthetic peptides fused to a fluorescent protein.
  • Binding properties were also assessed by applying synthetic peptides fused to biotin to live HEK-293T cells transfected for the extracellular domain of LRRC37B (versus an empty vector): they showed a binding of ExonS 40-63 (affinity of 0.4nM) and ExonS (affinity of 4nM) but not ExonS 4-23 (Figure 3H), said ExonS 4-23 corresponding to a peptide of SEQ. ID No. 16. To conclude, this shows that LRRC37B binds to the ExonS 40-63 amino acids of FGF13A.
  • Extracellular FGF13A decreases neuronal excitability by binding to NAV channels, which is enhanced by LRRC37B
  • Example 5 LRRC37B binds to NAV beta subunit 1 (SCN1B) of the voltage-gated channel through its B- specific domain
  • Example 6 LRRC37B regulates human cortical neuron excitability.
  • LRRC37B, FGF13A, SCN1B and SCN8A were co-expressed in human cortical neurons.
  • Coimmunoprecipitation experiments from human cortex tissue confirmed that LRRC37B, SCN1B, and FGF13A are present in the same complex ( Figure 6A).
  • LRRC37B+ neurons display less excitability than LRRC37B- neurons, with a decreased AP firing rate (Figure 6B(a), 6B(b)), an increased rheobase (Figure 6C) and AP risetime (Figure 6D) and width ( Figure 6C-E).
  • Genome and transcriptome analysis Encoding genes paralogs and orthologs originated from (Giannuzzi et al., 2012) and Ensembl gene trees (https://www.ensembl.org). Transcriptomic comparison between species is an analysis of data from Henrik Kaessmann laboratory (Heidelberg, Germany) described in (Cardoso-Moreira et al., 2020) and available at https://apps.kaessmannlab.org/evodevoapp/.
  • ENSG00000176681 human LRRC37A
  • ENSG00000238083 human LRRC37A2
  • ENSG00000176809 human LRRC37A3
  • ENSG00000185158 human LRRC37B
  • ENSMMUG00000008199 macaque LRRC37-M1
  • ENSMMUG00000063877/ ENSMMUG00000011880 macaque LRRC37-M2
  • ENSMMUG00000018463 macaque LRRC37-M7
  • ENSMUSG00000078632 mouse LRRC37A
  • ENSMUSG00000034239 mouse GM884.
  • LRRC37B cDNA originates from IRCMp5012D0514D (SourceBiosciences) whose sequence miss ExonS 4-5 which have been amplified by PCR from a cDNA library derived from GW18 fetal cortex (Suzuki et al., 2018) using the primers designed on the basis of the sequence of reference genome. The size of PCR fragment was confirmed and PCR fragment was subcloned into the Bsmbl and Ecorl restriction sites of the original cDNA by In Fusion cloning.
  • LRRC37B cDNA has been inserted by PCR amplification and InFusion cloning into the multicloning site between CAG promotor and IRES in the lentiviral backbone pCIG (CAG-IRES-EGFP-WPRE, Addgene #122953) (Suzuki et al., 2018) and pCIG-LSL (CAG-LSL-IRES-EGFP-WPRE) described in (Iwata et al., 2020). Resulting pCIG-LRRC37B and pCIG-LSL- LRRC37B plasmids have been used compared to pCIG and p-CIG-LSL in in utero electroporation experiments.
  • LRRC37B cDNA has been PCR amplified adding a Cter HA tag and inserted into the pCIG backbone at the multicloning site between CAG promotor and IRES. Resulting pCIG-LRRC37B-HA plasmid has been used compared to pCIG in in utero electroporation experiments. All constructs were verified by DNA sequencing. Predicted extracellular sequence lacking the signal peptide of LRRC37B (LRRC37BECTO) corresponding cDNA has been cloned by PCR amplification into a modified pCMV6-XL4 as described in (Apostolo et al., 2020) leading to pLRRC37B-Fc using InFusion cloning.
  • Fc-fusion protein contain a prolactin leader peptide (PLP) followed by an N-terminal FLAG tag, ectodomain of interest, a 3CPro cleavage site, and the dimeric human Fc domain.
  • PLP prolactin leader peptide
  • AP LRRC37B-alkaline phosphatase
  • FGF13A cDNA plasmid originates from Origene (RC204164) and has been PCR amplified for insertion into pCIG plasmid by InFusion cloning.
  • FGF13B, FGF13VY, FGF13core have been PCR amplified from FGF13A cDNA with primers targeting the core domain of FGF13 and with ExonS B or VY in the 5' primer. All constructs were verified by DNA sequencing.
  • pCMV-macaqueLRRC37B-HA ORF XP_028692824.1
  • pCMVchimpanzeeLRRC37B-HA ORF CK820_G0028539
  • pCAG-GEPH.FingRtdTomato-IL2RGTC is a gift from Juan Burrone (United Kingdom), derived from pCAG_GPHN.FingR-mKate2-IL2RGTC (Addgene #46297) described in (Gross et al., 2013).
  • pGPR158_ECTO, pSLIRTK2_ECTO and pLRRTMl_ECTO were previously described (Condomitti et al., 2018; Schroeder et al., 2018).
  • pCD4_ECTO is a gift from Lui's Ribeiro (Joris de Wit's laboratory), with the cDNA of the predicted extracellular domain of CD4, originating from pCMV-CD4 (Addgene #51604) described in (Raissi et al., 2013), inserted into the pDisplayTM Mammalian Expression Vector (ThermoFisher V66020).
  • HEK-293T cells Live staining of HEK-293T cells.
  • HEK-293T cells were split on coverslips and have been transfected with 500ng of each construct total amount (lug) of DNA (pCIG + pCIG-humanLRRC37B-HA or + pCMVchimpanzeeLRRC37B-HA or + pCMV-macaqueLRRC37B-HA or + pCIGhumanLRRC37A2-HA), using XtremeGene9 transfection reagent.
  • lug total amount of DNA
  • XtremeGene9 transfection reagent For live staining, 48 hours after transfection, medium was washed with cold PBS and then rabbit anti-LRRC37B (1:1000, as described above) was applied in PBS for lh at 4°C.
  • pCIG-LSL plasmids 1000 ng/pL were used together with pCAG-cre (15 ng/ pL) eventually with pCAG-GEPH.FingR-tdTomato-IL2RGTC while for electrophysiology pCIG plasmids were used (1 pg/pl) (see above).
  • the embryo's head was then placed between the paddles of pair of tweezer electrodes with the cathode lateral to the filled ventricle and five 50 ms, 30 V pulses were delivered with an interval of 950ms by a BTX830 electroporator (Harvard Apparatus). After electroporation, the uterus was replaced, the incision sutured closed and placed on a heating plate until recovery.
  • Mouse cortex processing and immunostaining Mouse P28 animals were perfused transcardiacally with ice-cold sucrose 8% PFA 4%. Brains were dissected and soaked in the same fixative for 3 hours, then stored in PBS azide. Then they either have been sectioned in 80 pm thickness using vibratome or 50 pm thickness using cryostat after dehydration in sucrose 30% and freezing in OCT. Slices were transferred into the blocking solution (PBS 0.3% Triton, 5% horse serum, 3% BSA) and incubated for 1 hour.
  • Brain floating slices were incubated 3 days at 4°C with primary antibodies: rabbit anti-LRRC37B (1:1000, as described above), mouse anti-ankyrin-G (1:500, as described above), mouse anti-FGF13A (1:500; MAS- 27705, ThermoFisher), chicken anti-EGFP (1:1000; abl3970, Abeam), rat anti-mCherry which recognizes tdTomato (1:1000; M11217, ThermoFisher), mouse anti-pan-NAVa (1:500; S8809, Merck).
  • VGAT staining For VGAT staining (guinea pig anti-VGAt 1:500; 131 004, Synaptic Systems), stainings have been done sequentially in blocking solution PBS 1% Triton, 5% horse serum, 3% BSA. After three PBS washes, slices were incubated overnight at 4°C with secondary antibodies in PBS: donkey anti-rabbit Cy3, anti-rabbit a594, antimouse a647, anti-chicken a488, anti-rat Cy3, anti-guinea pig a647 (1:1000 or 1:250 for cryosections used for STED imaging) and Hoechst (1:10000). After three washes in PBS, brain sections were mounted on a slide glass with the mounting reagent (DAKO glycerol mounting medium) using #1.5 coverslips.
  • DAKO glycerol mounting medium the mounting reagent
  • Image acquisition Confocal images were obtained with Zeiss LSM880 and LSM900 driven by Zen Black and Blue software equipped with objectives lOx, 20x, oil immersion 25x and oil immersion 40x, AiryScan system and argon, helium-neon and 405 nm diode lasers.
  • STED single focal section images were obtained with an Abberior system with Olympus 1X83 body equipped with lOOx oil immersion, 480, 532, 640 nm excitation lasers and 595nm 775 nm depletion lasers.
  • STED pictures were deconvoluted using Huygens deconvolution software. Except if specified, representative pictures are maximum projections.
  • AIS intensity profile was done on maximum projection pictures in Matlab as described in (Grubb and Burrone, 2010).
  • mouse the beginning of the AIS was set using the EGFP channel (starting from the soma).
  • ankyrin-G was used to set the beginning of the axon. Puncta and area quantification have been done using Fiji.
  • 10 focal sections (0.4 pm thickness) maximum projection was used for quantification.
  • STED imaging one focal section was used only with on average 10pm length in the proximal part of the AIS.
  • EGFP was used to delineated the soma and the AIS (30pm starting from the soma). Binarized pictures of the gephyrin-tdTomato, VGAT, FGF13A have been used to quantify manually the number of puncta or their positive area.
  • Electrophysiological recordings For mouse experiments, coronal slices were prepared from postnatal day P24-32 animals. Briefly, after decapitation, the brain was quickly removed and transferred into ice- cold cutting solution (in mM): 87 NaCI, 2.5 KCI, 1.25 NaH2PO4, 10 glucose, 25 NaHCO3, 0.5 CaCI2, 7 MgCI2, 75 sucrose, 1 kynurenic acid, 5 ascorbic acid, 3 pyruvic acid, pH 7.4 with 5% CO2/95% 02, and whole brain coronal slices (250 pm) were cut using a vibratome (VT1200, Leica Biosystems). Afterward, slices were transferred to 32 °C cutting solution for 45 min to recover and finally maintained at room temperature until used for recordings.
  • ice- cold cutting solution in mM
  • Slicing solution contained choline chloride HOmM, NaHCO3 26mM, Na- ascorbate 11.6 mM, Dglucose lOmM, MgCI27mM, Na-pyruvate 3.1 mM, KCI 2.5 mM, NaH2PO4 1.25mM, CaCI2 0.5 mM; 300-315 mOsm, pH adjusted to 7.4, with 5% CO2/95% 02 and was ice-cold.
  • Recovery solution was the same than the ACSF used for the transfer from hospital. Slicing was performed with a vibrating blade microtome or using a comprestome, and 300-pm slices were incubated for around 30 min at 32 °C in ACSF.
  • mice and human brain slices were continuously perfused (32-34°C) in a submerged chamber (Warner Instruments) at a rate of 3-4 ml/min with (in mM): 127 NaCI, 2.5 KCI, 1.25 NaH2PO4, 25 NaHCO3, 1 MgCI2, 2 CaCI2, 25 glucose at pH 7.4 with 5% CO2/95% 02.
  • Whole-cell patch-clamp recordings were done using borosilicate glass recording pipettes (resistance 3.5-5 MO, Sutter P-1000), using a double EPC-10 amplifier under control of Patchmaster v2 x 32 software (HEKA Elektronik, Lambrecht/Pfalz, Germany).
  • the following internal medium was used (in mM): 135 K-Gluconate, 4 KCI, 10 HEPES, 4 Mg-ATP, 0.3 Na2-GTP, 10 Na2- phosphocreatine, 3 biocytin (pH 7.3).
  • Cell intrinsic properties were recorded in current clamp, while sEPSCs and sIPSCs were recorded in voltage clamp at -70 mV and 0 mV, respectively. Currents were recorded at 20 Hz and low-pass filtered at 3 kHz when stored. The series resistance was compensated to 75-85%.
  • Cell intrinsic properties were analyzed using Fitmaster (HEKA Elektronik, Lambrecht/Pfalz, Germany), spontaneous input was analyzed using Mini Analysis program (Synaptosoft).
  • Phase plot analysis compares the rate of change (first derivative) in voltage during APs (y-axis) to the membrane voltage (x- axis).
  • the first current step (1 sec) to initiate AP(s) was used for phase plot analysis.
  • the AIS, somatic and repolarization voltages were determined as the absolute membrane potential measured at the respective peaks.
  • Elisa assay An ELISA-based assay (Ozgul et al., 2019) was used to identify the interaction between ectodomain (as defined in the Uniprot database) of 920 cell surface or secreted proteins cloned in frame with an Fc domain against LRRC37B-ECTO-AP fusion as described in (Apostolo et al., 2020). Horseradish peroxidase (HRP) conjugated anti-Fc antibody develops a blue colour if the prey (FGF13A) remain bound to the bait (LRRC37B) after the washes. After the initial identification, the experiment has been repeated 3 times using triplicate wells.
  • HRP horseradish peroxidase
  • the library contained AIS proteins or proteins coded by genes enriched in chandelier interneurons as described in (Bakken et al., 2021; Favuzzi et al., 2019; Leterrier, 2016): ALCAM, CDH4, CDH6, CDH11, CNTNAP5, DPP10, FGF13 isoform 1 (FGF13A), FSTL5, ITGAV, ITGA6, LRRN1, LRRN2, NFASC, OLFM3, PCDH19, PCSK2, ROBO1, SGCD, SLITRK1, SLITRK5, TENM4, THSD7A, UNC5B. They were all negative except FGF13A.
  • HTK293T have been transfected with 500-2000ng of DNA using XtremeGene9 transfection reagent. 72 hours after transfection, cells were lysed in lysis buffer (50mM Tris-HCI, pH 7.5, 1% sodium deoxycholate, 0.1% Triton, proteinase inhibitors) on a wheel for one hour in a cold room. When required, FGF13A and its ExonS where applied in the culture medium 5 hours before protein extraction. EGFP positive area of P17 mouse cortex has been dissected using forceps in cold PBS of brains after cervical dislocation.
  • lysis buffer 50mM Tris-HCI, pH 7.5, 1% sodium deoxycholate, 0.1% Triton, proteinase inhibitors
  • Cortices have been homogenized in homogenization buffer (50mM Tris-HCI, pH 7.5, 1% sodium deoxycholate, proteinase inhibitor) using a Dounce homogenizer. After Triton addition (0.1% final concentration), samples have been rotated on a wheel for one hour in a cold room.
  • Human cortices (from 14yo-48yo patients) have been homogenized in homogenization buffer (50mM Tris-HCI, pH 7.5, 1% sodium deoxycholate, proteinase inhibitor) using a Dounce homogenizer. After Triton addition (0.1% final concentration), samples have been rotated on a wheel for one hour in a cold room.
  • samples were centrifugated 25mn 16000g and lysates transferred in a new tube with addition of NaCI (final concentration 150 mM).
  • Samples were incubated overnight on a wheel in a cold room with HA magnetic beads or protein A magnetic beads coupled with lug of mouse anti-pan-NAVa (as described above) or rabbit anti-FLAG (abll62, Abeam) or mouse anti- FGF13A (as described above) or mouse IgG (ThermoFisher) antibodies. Beads were washed 4 times with the washing solution (50mM Tris-HCI, pH 7.5, 1% sodium deoxycholate, 0.1% Triton, 150 mM) and one time with PBS.
  • the washing solution 50mM Tris-HCI, pH 7.5, 1% sodium deoxycholate, 0.1% Triton, 150 mM
  • samples were eluted in 2x Laemmli buffer at 95°C.
  • Medium was centrifugated 25mn 16000g and diluted to final lx Laemmli buffer at 95°C.
  • the input (in lx Laemmli buffer), medium, and immunoprecipitated samples were run in NUPAGE 12% Bis-Tris Protein Gel at the voltage of 90V for 2 hours in MOPS buffer and then transferred to PVDF Blotting Membrane at the voltage of 100V for 100 minutes.
  • the membrane was blocked in the buffer (5% skim milk and 0.1% Tween20 in TBS) for 1 hour at room temperature and subsequently incubated in the blocking buffer containing rabbit anti-HA (1:1000, as described above), rabbit antiLRRC37B (1:1000, as described above), mouse anti-FGF13A (1:1000, as described above), mouse anti-pan-FGF13 (1:1000; PA5-27302, ThermoFisher), mouse antibeta-actin (1:5000; MAI-140, ThermoFisher), mouse anti-pan-NAVa (1:500, as described above), rabbit anti-FLAG (1:1000; as described above), rabbit anti-SCNIB (1:500; CST 13950S, Bioke) antibodies overnight at 4°C, followed by the incubation in the blocking solution containing secondary antibody anti-Rabbit or Mouse IgG antibody conjugated with HRP at room temperature for 1 hour. Pierce ECL Western Blotting Substrate was used for signal detection.
  • Binding assay & affinity approximation To estimate the FGF13A - LRRC37B affinity on the cell surface, a binding assay approach has been used as described in (Savas et al., 2015). Briefly, HEK-293T cells were plated on 10cm plates, non-transfected (6 times) or transfected with the pdisplay empty vector (6 times), LRRC37B_ECTO (6 times), LRR_ECTO (3 times) ALRR_ECTO (3 times)(described above), cultured for 24 hours, gently trypsinized and re-plated on 24 well plates and cultured for an additional 24 hours.
  • Live cells were incubated with FGF13A ExonS-biotin at 0, 1, 5, 10, 50, 100 nM, fixed, probed with a streptavidin-HRP and reacted with TMB. The reaction was stopped with IN HCI and transferred to 96 well plates and the absorbance was measured on a plate reader at 450nm. All saturation binding calculations were performed with GraphPad Prism, One site -Specific binding, non-linear fit curve.
  • LRRC37B (aa 28-905, containing the entire ectodomain) ectoFC protein was produced by transient transfection of HEK293T cells using PEI (Polysciences). Six hours after transfection, media was changed to OptiMEM (Invitrogen) and harvested 5 days later. Conditioned media was centrifuged, sterile filtered and run over a fast-flow Protein-G agarose (Thermo-Fisher) column.
  • Crude synaptosome extracts were prepared from 2-3 P21-22 rat brains per condition, homogenized in homogenization buffer (4 mM HEPES pH 7.4, 0.32 M sucrose and protease inhibitors) using a Dounce homogenizer. Homogenate was spun at 1,000 x g for 10 minutes at 4°C. Supernatant was spun at 14,000 x g for 20 minutes at 4°C.
  • homogenization buffer 4 mM HEPES pH 7.4, 0.32 M sucrose and protease inhibitors
  • Beads were packed into Polyprep chromatography columns (BioRad) and washed with 50 mL of high-salt wash buffer (50 mM HEPES pH 7.4, 300 mM NaCI, 0.1 mM CaCI2, 5% glycerol and protease inhibitors), followed by a wash with 10 mL low-salt wash buffer (50 mM HEPES pH 7.4, 150 mM NaCI, 0.1 mM CaCI2, 5% glycerol and protease inhibitors). Bound proteins were eluted from the beads by incubation with Pierce elution buffer and TCAprecipitated overnight.
  • high-salt wash buffer 50 mM HEPES pH 7.4, 300 mM NaCI, 0.1 mM CaCI2, 5% glycerol and protease inhibitors
  • 10 mL low-salt wash buffer 50 mM HEPES pH 7.4, 150 mM NaCI, 0.1 mM CaCI2, 5% gly
  • the precipitate was re-suspended in 8 M Urea with ProteaseMax (Promega) per the manufacturer's instruction.
  • the samples were subsequently reduced by 20-minute incubation with 5mM TCEPO (tris(2carboxyethyl)phosphine) at RT and alkylated in the dark by treatment with 10 mM lodoacetamide for 20 additional minutes.
  • TCEPO tris(2carboxyethyl)phosphine
  • the proteins were digested overnight at 37°C with Sequencing Grade Modified Trypsin (Promega) and the reaction was stopped by acidification. Mass spectrometry analysis was performed by the VIB Proteomics Core (Ghent, Belgium).
  • experiments with HEK-293T cells have been performed >3 times, experiments from mouse in vivo samples > 2 times and human in vivo samples >3 times.
  • human AIS intensity profile data we analysed 60 neurons per patient, 3 patients (7-38 years old); and for electrophysiology, 4-6 neurons per patient and 7 patients (4- 62 years old).

Abstract

The application concerns a novel hominid-specific pathway to modulate neuronal excitability. In a first aspect, the application relates to a modulator of the interaction between a LRRC37B protein and the NAV1.6 voltage-gated sodium channel for use as a medicine. Furthermore, means and methods are provided to treat neurological disorders characterized by affected neuron excitability. In further aspects, the invention relates to methods for identification of or for screening for modulators of neuronal excitability.

Description

MEANS AND METHODS TO MODULATE NEURON EXCITABILITY
FIELD OF THE INVENTION
The invention relates to the field of neurological diseases. More particularly, the application discloses a novel hominid-specific pathway to modulate neuronal excitability. Means and methods are provided to treat neurological disorders characterized by affected neuron excitability. The invention further teaches methods for identification of or for screening for modulators of neuronal excitability.
BACKGROUND OF THE INVENTION
Information processing in neurons relies on the integration of excitatory and inhibitory inputs to make a yes-or-no decision to fire an action potential (AP). AP occurs in an all-or-none fashion as a result of local changes in membrane potential brought about by net positive inward ion fluxes. They occur due to depolarization of the neuronal membrane, with membrane depolarization propagating down the axon to induce neurotransmitter release at the axon terminal. The input (synaptic inputs) - output (AP firing rate) ratio determines neuronal excitability. In most neurons this regulation of neuronal excitability occurs at several neuronal compartments including the axon initial segment (AIS), a specialized domain of the axon proximal to the soma, clustering and maintaining ion channels in high densities including the voltage-gated sodium channel NAV1.6/SCN8A.
Neuron excitability is a precisely controlled mechanism and disruption of the balance between excitation and inhibition lies at the basis of several neurological disorders. Most known examples of a hyperexcitable neuron state are seizures, epilepsy, epileptogenesis and pain. It would thus be advantageous to develop methods to modulate neuronal excitability.
SUMMARY
Here the Applicants disclose a novel therapeutic avenue to modulate neuronal excitability. More particularly, it is shown that the LRRC37 family of hominid-specific genes, which encode orphan Leucine- Rich-Repeat (LRR) receptors, are expressed as a unique repertoire in human cortical neurons. Among these, LRRC37B protein, further in the text referred to as LRRC37B, is a receptor displayed selectively at the level of the axon initial segment (AIS) of human cortical pyramidal neurons. It was surprisingly found that LRRC37B overexpression in vivo lead to reduced intrinsic neuron excitability. Furthermore, LRRC37B was found to act through direct binding to secreted FGF13A ligand, leading to inhibition of the voltagegated sodium channel a (alpha) subunit named NAVI.6 (encoded by the SCN8A gene), the main voltagegated sodium channel responsible to AP generation. Said FGF13A ligand is referred to as FGF13A protein or FGF13A, in the description below. LRRC37B also binds to the regulatory SCN1B beta-subunit of the PVDH/LRRC37/769 voltage-gated sodium channel, and thereby negatively regulates SCN1B interaction with the alpha subunit, known as NAVI.6. Ex vivo physiological experiments on adult human cortical tissue revealed reduced excitability in pyramidal neurons expressing LRRC37B, indicating its physiological relevance for human neuronal function. The data presented herein identify LRRC37B as a human-specific modifier of cortical neuron excitability that acts through interactions with FGF13A and/or SCNB1, preferably acts through interactions with FGF13A and SCNB1. The novel modulators and/or modulation mechanisms disclosed herein with important implications, may allow for better understanding of brain function, evolution, and/or provide for new agents, means and/or methods for use in excitability disorders such as seizures and epilepsy.
Based on the data herein disclosed, it is a first aspect of the application to provide modulators of the LRRC37B-FGF13A-SCN1B complex for use as a medicine, more particularly for modulating neuronal excitability, even more particularly for treating neuronal excitability disorders such as seizures and epilepsy. In a first aspect, a modulator of the interaction between the LRRC37B protein or receptor and the NAVI.6 voltage-gated sodium channel for use as a medicine is provided. In a first embodiment, said modulator is a modulator of the LRRC37B-FGF13A interaction and/or of the LRRC37B-SCN1B interaction, preferably LRRC37B-FGF13A interaction or of the LRRC37B-SCN1B interaction. The application also provides an isolated FGF13A protein fragment, preferably said isolated FGF13A fragment comprising or consisting of an amino acid sequence as depicted in SEQ ID No. 4, more preferably said isolated FGF13A fragment comprising or consisting of an amino acid sequence SEQ ID No. 15 for therapeutic application and/or for screening purposes. Furthermore, the application also provides the isolated LRRC37B protein fragment comprising or consisting of the amino acid sequence as depicted in SEQ ID No. 8 or in SEQ ID No. 12 for therapeutic applications or and/or for screening purposes.
The application also provides a modulator of the interaction between LRRC37B and the NAVI.6 voltagegated sodium channel for use as a medicine. In a particular embodiment, said modulator is a peptide comprising a fragment of FGF13A, said fragment of FGF13A preferably being FGF13A S-fragment as depicted in SEQ ID No. 4 or a peptidomimetic thereof. In a preferred embodiment, said modulator is a peptide comprising the fragment of FGF13A comprising or consisting of SEQ ID No. 15 or a peptidomimetic thereof.
In a second aspect, the application provides methods of identifying modulators of neuronal excitability, more particularly modulators of the LRRC37B-FGF13A-SCN1B complex, even more particularly modulators of the LRRC37B-FGF13A interaction and/or of the LRRC37B-SCN1B interaction, preferably modulators of the LRRC37B-FGF13A interaction or of the LRRC37B-SCN1B interaction. Said methods comprise the steps of contacting the LRRC37B protein or a fragment thereof with the FGF13A protein or a fragment thereof and/or the SCN1B protein or fragment thereof in the presence or absence of a test compound and identifying said test compound as modulator of neuronal excitability and function if said test compound is statistically significantly reducing or increasing the binding of the LRRC37B protein or fragment thereof with the FGF13A protein and/or with the SCN1B protein or fragments thereof, compared to identical conditions but in absence of the test compound. In one embodiment, the binding between the protein partners can be determined, for example, by immunologic or radiologic detection, co-sedimentation, co-immunoprecipitation or electron microscopy. In another embodiment, the LRRC37B or the fragment thereof can be provided by cells expressing LRCC37B or the fragment thereof. In yet another embodiment, the FGF13A or the fragment thereof can be administered extracellularly to the cells expressing LRRC37B or the fragment thereof. In another embodiment, said test compound is a peptide consisting of less than 20 contiguous amino acids and is a fragment of the LRRC37B-specific domain as, for example, depicted in SEQ ID No. 12. In another embodiment, said test compound is, for example, a peptidomimetic of said peptide.
The application also provides a method of identifying a modulator of neuron excitability comprising the steps of generating a peptide consisting of less than 20 contiguous amino acids wherein the peptide is a fragment of the FGF13A S-fragment, herein also referred to as FGF13A S-domain as depicted in SEQ ID No. 4 or SEQ ID No. 15 or generating a peptidomimetic of said peptide, and contacting one or more of the peptides or peptidomimetics with the NAVI.6 voltage-gated sodium channel as depicted in SEQ ID No. 14 or a functional fragment thereof, and identifying as a modulator of neuron excitability a peptide or peptidomimetic that, compared to identical conditions but in absence of the peptide or peptidomimetic, statistically significantly reduces or increases the activity of NAVI.6. In one embodiment, NAVI.6 or the functional fragment thereof is provided by cells expressing NAVI.6 or the functional fragment thereof. In a particular embodiment, said cells are electrically excitable cells, such as neurons or cardiac cells.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1A shows the LRRC37 transcripts detected in the cerebral cortex in mouse, macaque and human. RPKM stands for reads per kilobase million. Figures 1B-C illustrate the proportion of LRRC37 positive cell types (B) and pyramidal neurons (C) in human postnatal cortical samples at different ages (yo: years old). RPKM stands for reads per kilobase million. Figure ID shows the expression of LRRC37 transcripts in cell types of human and chimpanzee prefrontal cerebral cortex. CPM stands for counts per million. Figure IE illustrates the expression of LRRC37 transcript in CPM during the postnatal human life (from neonates to adulthood) in the prefrontal cerebral cortex. Figure 2A-B show that LRRC37B colocalizes with ankyrin-G in a subset of human neurons from individuals older than 2yo with no differences in the AIS length of LRRC37B+ neurons compared to LRRC37B- neurons (Mann-Whitney test). Figure 2C shows the immunodetection of LRRC37B in the mouse cerebral cortex transfected with LRRC37B cDNA and colocalization with ankyrin-G. Figure 2Da-b and E show that LRRC37B mouse transfected neurons have a decreased neuronal excitability compared to control neurons transfected with EGFP cDNA only (control): the LRRC37B positive cells display a decreased action potential (AP) firing rate (2-way ANOVA test) (D a-b), an increased rheobase (E, left) and an increase AP risetime (E, center) and width (E, right) (Mann-Whitney test). Figure 2F illustrates that LRRC37B is detected in a subset of human cortical AIS from individuals older than 2 year old to reach a plateau from 10 years old. ns, non-significative; *, p<0.05; **, p<0.01; ***, p<0.001.
In Figure 3 herein below, the FGF13A S-fragment as depicted in SEQ ID No. 4 is referred to as "ExonS". Figure 3A is an illustration of the several FGF13 spliced isoforms (upper panel) and shows that FGF13A is the only secreted FGF13 (lower panel). Figure 3B shows that LRRC37B binds only to FGF13A as demonstrated by LRRC37B-HA immunoprecipitation from co-transfected HEK-293 cells with cDNAs coding for FGF13A, FGF13B, FGF13VY and the core domain of FGF13 proteins. Figure 3C shows the coimmunoprecipitation with LRRC37B-HA of recombinant FGF13A and its synthetic ExonS applied in the culture medium of HEK-293 cells transfected with LRRC37B-HA cDNA. Figure 3D shows that FGF13A co- immunoprecipitates in transfected HEK-293 cells with the LRRC37B extracellular part (LRRC37Bectoml) as well as with its leucin-rich repeats (LRR) (LRRC37Bectom2) but not with the extracellular part devoid of the LRR (LRRC37Becto). Figure 3E is an illustration of the different LRRC37B protein fragments tested. Figure 3F depicts a peptide array experiment with LRR protein application on 10 amino acid peptides windows covering the ExonS sequence which enables to identify two possible binding sites: ExonS amino acids 4-23 (as depicted in SEQ ID No. 16, and herein referred to as "ExonS 4-23") and ExonS amino acids 40-63 (as depicted in SEQ ID No. 15, and herein referred to as "ExonS 40-63"). Figure 3G shows fluorescent polarization (FP) assay between LRR protein (different doses) and ExonS of SEQ ID No. 4 (marked as "ExonS"), and also fragments of ExonS: ExonS 40-63, depicted in SEQ ID No. 15 (marked as "Exon S 40-63"), ExonU (marked as "FGF13B ExonU") and two Random peptides as defined in Figure 3F; LRR binds to the ExonS at an affinity of 530nM and ExonS 40-63 (i.e. the peptide of a sequence as depicted in SEQ ID No. 15) at an affinity of 160nM. Figure 3H shows affinity estimation binding assays with peptide application of ExonS or fragments thereof (the fragment as depicted in SEQ ID No. 16, herein annotated as "ExonS 4-23"; the fragment as depicted in SEQ ID No. 15 herein annotated as "ExonS 40-63" or the FGF13A S-fragment as depicted in SEQ ID No. 4, herein annotated as "ExonS") on HEK-293T cells transfected for LRRC37B_ECTO (as defined in panel D-E) or empty vector: only ExonS 40-63 (affinity 0.4nM) and ExonS (affinity 4nM) bind to LRRC37B. In Figure 4 herein below, the FGF13A S-fragment as depicted in SEQ. ID No. 4 is referred to as "ExonS", while shorter peptide fragment consisting of amino acids from position 40 to 63, as depicted in Seq ID No. 15 is referred to as "ExonS 40-63". Figure 4A(a)-B(a) show that extracellularly applied recombinant FGF13A on mouse cortical sections decreases neuronal excitability (A), AP firing rate (B left, 2-way ANOVA test and paired Wilcoxon test) and rheobase (B right, paired Wilcoxon test). Figure 4A(b) shows that extracellularly applied recombinant FGF13A, synthetic ExonS, ExonS 40-63 on mouse cortical sections decreases neuronal excitability when compared to ExonS 1-39 (consisting of aa from position 1 to position 39 of ExonS sequence). Figure 4B(b) shows normalization (to values before application for each neuron) of AP firing rate (left, paired Wilcoxon tests) and rheobase (right, paired Wilcoxon test). Figure 4C(a) shows that extracellular application of the FGF13A S fragment on mouse cortical sections is sufficient to decrease neuronal excitability (left) in contrast to intracellular application of said FGF13A S fragment (right). Figure 4C(b) shows that intracellular application of FGF13A does not lead to decreased neuronal excitability (right, Mann-Whitney test). Figure D(a)-G(a) shows the effect of extracellular application of FGF13A (D(a)-E(a)) and the FGF13A S fragment (F(a)-G(a)) on Na (D(a)-F(a)) and K (E(a)- G(a)) currents. Figure 4D(b)-E(b) shows the effect of extracellular application of FGF13A, ExonS, ExonS 40-63 but not ExonS 1-39 on Na (D(b), right normalization to values before application for each neuron, paired Wilcoxon tests) and K currents (E(b), right normalization to values before application for each neuron, paired Wilcoxon tests). Legend: ns, non-significant; *, p<0.05; **, p<0.01; ****, p<0.0001. Figure 4H shows the NAVI.6 immunoprecipitations from HEK-293 cells transfected for NAVI.6 and FGF13A with or without LRRC37B, more particularly NAVI.6 binds FGF13A and that they form a complex with LRRC37B. Figure 41 shows the co-immunoprecipitation of FGF13A with LRRC37B-HA from cortical extracts (P17) of LRRC37B-HA/EGFP transfected mice compared to control. Figures 4J-M show that LRRC37B co-localizes with FGF13A and NAVa subunits based on STED microscopy of the AIS of mouse neurons transfected for LRRC37B (J) and that FGF13A abundance increases (K) at the AIS (L) upon LRRC37B expression in contrast to NAVa subunits (M).
Figure 5A shows the SCN1B immunoprecipitations from HEK-293 cells transfected for SCN1B +/-, LRRC37B +/- and FGF13A +/- cDNAs. Figure 5B is an illustration of the LRRC37B deletion mutants. Figure 5C shows that SCNB1 co-immunoprecipitates with the LRRC37B extracellular part as well as its specific LRRC37B domain but not with the extracellular part devoid of the specific B domain in transfected HEK- 293 cells. Figure 5D shows the NAVI.6 immunoprecipitations from HEK-293 cells transfected for NAVI.6, SCN1B and LRRC37B +/- cDNAs. Figure 5E shows the immunoprecipitation of the extracellular part of SCN1B with a synthetic peptide corresponding to the LRRC37B specific domain (LB133-186) from HEK- 293 cells transfected for the cDNA coding for the extracellular part of SCN1B. Figure 5F shows the SCNB1 and NAVI.6 immunoprecipitations from HEK-293 cells transfected for NAVI.6, SCN1B, with or without LB133-186.
Figure 6A(a) shows that LRRC37B forms a complex with FGF13A and SCN1B in the human cortex. Figure 6A(b) shows that LRRC37B forms a complex with FGF13A, SCN1B and NAVa subunits (i.e. NAV 1.6) in the human cortex. Figures 6B(a)-E(a) show that LRRC37B+ neurons display a decreased excitability compared to LRRC37B- neurons (2-way ANOVA test for the AP firing rate, Mann-Whitney test for other parameters). *, p<0.05; ****, p<0.0001. Figures 6B(b) and F show that LRRC37B+ neurons display a decreased excitability compared to LRRC37B- neurons (Mann-Whitney tests). Legend: ns, nonsignificant; *, p<0.05; ***, p<0.001; ****, p<0.0001.
Figure 7 is an illustration showing the proposed model on how the LRRC37B-FGF13A-SCN1B complex acts on the NAVI.6 voltage-gate sodium channel to modulate neuron excitability in the human cortex. The FGF13A S-fragment of the FGF13A as the extracellular ligand, is indicated as the darker part of FGF13A rectangle.
DETAILED DESCRIPTION
Definitions
In order that the present description can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description. The present invention is described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence", is understood to represent one or more nucleotide sequences. As such, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
It is understood that wherever aspects or embodiments are described herein with the language "comprising", otherwise analogous aspects or embodiments described in terms of "consisting of" and/or "consisting essentially of" are also provided. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., current Protocols in Molecular Biology (Supplement 100), John Wiley & Sons, New York (2012), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.
Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.
The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). For example, if it is stated that a modulator of the LRRC37B-FGF13A-SCN1B interaction reduces the activity or function of NAVI.6 in a cell by at least about 60%, it is implied that the NAVI.6 activity or function is reduced by a range of 50% to 70%.
As used herein, the terms "nucleic acid", "nucleic acid sequence" or "nucleic acid molecule" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acids may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of nucleic acids include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular. The nucleic acid may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5' or 3' untranslated regions, a reporter gene, a selectable marker or the like. The nucleic acid may comprise single stranded or double stranded DNA or RNA. The nucleic acid may comprise modified bases or a modified backbone. A nucleic acid that is up to about 100 nucleotides in length, is often also referred to as an oligonucleotide. "Nucleotides" as used herein refer to the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which are absent in nucleosides). A nucleotide without a phosphate group is called a "nucleoside" and is thus a compound comprising a nucleobase moiety and a sugar moiety. As used herein, "nucleobase" means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Naturally occurring nucleobases of RNA or DNA comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
"Amino acids" as used herein refer to the structural units (monomers) that make up proteins. They join together to form short polymer chains called peptides or longer chains called either polypeptides or proteins. These chains are linear and unbranched, with each amino acid residue within the chain attached to two neighbouring amino acids. Twenty amino acids encoded by the universal genetic code are naturally incorporated into polypeptides and are called proteinogenic or natural amino acids. Natural amino acids or naturally occurring amino acids are glycine (Gly or G), Alanine (Ala or A), Valine (Vai or V), Leucine (Leu or L), Isoleucine (He or I), Methionine (Met or M), Proline (Pro or P), Phenylalanine (Phe or F), Tryptophan (Trp or W), Serine (Ser or S), Threonine (Thr or T), Asparagine (Asn or N), Glutamine (Gin or Q), Tyrosine (Tyr or Y), Cysteine (Cys or C), Lysine (Lys or K), Arginine (Arg or R), Histidine (His or H), Aspartic Acid (Asp or D) and Glutamic Acid (Glu or E).
The terms "identical" or percent "identity" in the context of two or more nucleic acid or amino acid sequences refer to two or more sequences that are the same or have a specified percentage of nucleotides or amino acid residues respectively that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of nucleotide or amino acid sequences.
The term "percent sequence identity" or "% sequence identity" or "percent identity" or "% identity" between two polynucleotide or polypeptide sequences refers to the number of identical matched positions shared by the sequences over a comparison window, taking into account additions or deletions (i.e. gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where an identical nucleotide or amino acid is presented in both the target and reference sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides or amino acids. Likewise, gaps presented in the reference sequence are not counted since target sequence nucleotides or amino acids are counted, not nucleotides or amino acids from the reference sequence.
One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified in Karlin et a!., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, and incorporated into the NBLAST and XBLAST programs (Altschul et a!., 1991, Nucleic Acids Res., 25:3389-3402). In certain aspects, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain aspects, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternative aspects, the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) can be used to determine the percent identity between two amino acid sequences (e.g., using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certain aspects, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM 120 with residue table, a gap length penalty of 12 and a gap penalty of 4. One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain aspects, the default parameters of the alignment software are used.
One skilled in the art will appreciate that the generation of a sequence alignment for the calculation of a percent sequence identity is not limited to binary sequence-sequence comparisons exclusively driven by primary sequence data. Sequence alignments can be derived from multiple sequence alignments. One suitable program to generate multiple sequence alignments is ClustalW2, available from www.clustal.org. Another suitable program is MUSCLE, available from www.drive5.com/muscle/. ClustalW2 and MUSCLE are alternatively available, e.g., from the EBI (European Bioinformatics Institute). In certain aspects, the percentage identity "X" of a first nucleotide sequence to a second nucleotide sequence is calculated as 100 x (Y/Z), where Y is the number of nucleotide residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence. Different regions within a single polynucleotide target sequence that align with a polynucleotide reference sequence can each have their own percent sequence identity. It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.
According to the present application, the degree of identity, between a given reference nucleotide sequence and a nucleotide sequence which is a homologue of said given nucleotide sequence will preferably be at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of identity is given preferably for a nucleic acid region which is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the entire length of the reference nucleic acid sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given preferably for at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or 200 nucleotides, preferably contiguous nucleotides. In a particular embodiment, the degree/percentage of similarity or identity is given for the entire length of the reference nucleic acid sequence.
The term "amino acid identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. According to the present application, the degree of identity, between a given reference amino acid sequence and an amino acid sequence which is a homologue of said given amino acid sequence will preferably be at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of identity is given preferably for an amino acid region which is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of identity is given preferably for at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or 200 amino acids, preferably contiguous amino acids. In a particular embodiment, the degree/percentage of similarity or identity is given for the entire length of the reference amino acid sequence.
"Homologue" or "homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
"Contiguous" as used herein means next or together in sequence, hence the contiguous nucleotides or amino acids are linked nucleotides or amino acids (i.e. no additional nucleotides or amino acids are present between those that are linked).
The term "defined by SEQ ID No. X" or "as depicted in SEQ ID No. X" as used herein refers to a biological sequence consisting of the sequence of amino acids or nucleotides given in the SEQ ID No. X. For instance, a protein defined in/by SEQ ID No. X consists of the amino acid sequence given in SEQ ID No. X. A further example is an amino acid sequence comprising SEQ ID No. X, which refers to an amino acid sequence longer than the amino acid sequence given in SEQ ID No. X but entirely comprising the amino acid sequence given in SEQ ID No. X (wherein the amino acid sequence given in SEQ ID No. X can be, for example, located N-terminally or C-terminally in the longer amino acid sequence, or can be embedded in the longer amino acid sequence), or to an amino acid sequence consisting of the amino acid sequence given in SEQ ID No. X.
"Compound" or "test compound" used in the screening methods of the present application is not limited to a specific type of a compound and means any chemical or biological compound, including simple or complex organic and inorganic molecules, peptides, peptidomimetics, proteins, antibodies, carbohydrates, nucleic acids or derivatives thereof. The term "compound" is used herein in the context of a "drug candidate compound" or a "candidate compound for lead optimization" in therapeutics, described as identified with the screening methods herein disclosed. As such, these compounds comprise organic or inorganic compounds, derived synthetically or from natural resources. The compounds include polynucleotides, lipids or hormone analogs that are characterized by low molecular weights. Other biopolymeric organic test compounds include small peptides or peptide-like molecules (peptidomimetics) comprising from about 2 to about 40 amino acids and larger polypeptides comprising from about 40 to about 500 amino acids, such as antibodies or antibody conjugates.
The term "small molecule compound", as used herein, refers to a low molecular weight (e.g., < 900 Da or 40 < 500 Da) organic compound. The compounds also include polynucleotides, lipids or hormone analogues that are characterized by low molecular weights. Other biopolymeric organic test compounds include small peptides or peptide-like molecules (peptidomimetics) comprising from about 2 to about 40 amino acids and larger polypeptides comprising from about 40 to about 500 amino acids, such as antibodies or antibody conjugates.
In the screening methods herein disclosed, entire compound libraries can be screened. "Compound libraries" are a large collection of stored compounds utilized for high throughput screening. Compounds in a compound library can have no relation to one another, or alternatively have a common characteristic. For example, a hypothetical compound library may contain all known compounds known to bind to a specific binding region. As would be understood by one skilled in the art, the methods of the application are not limited to the types of compound libraries screened. For high-content screening, compound libraries may be used. Examples include, but are not limited to, natural compound libraries, allosteric compound libraries, peptide libraries, antibody fragment libraries, synthetic compound libraries, etc. In one embodiment, high throughput screening methods involve providing a library containing a large number of compounds (candidate compounds) potentially having the desired activity. Such "combinatorial chemical libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds identified can serve as conventional "hit compounds" or can themselves be used as potential or actual therapeutics. Test compounds can be selected from a collection of compounds, more particularly a library of biological and/or chemical compounds. Nonlimiting examples of said library can be a library comprising small molecules, compounds with known functions, FDA approved drugs, compounds pre-screened on bioactivity or can be a drug repurposing library.
In some instances, the screening methods herein described are "high content screening" (HCS) methods. Typically, HCS is an automated system to enhance the throughput of the screening process. However, the present invention is not limited to the speed or automation of the screening process. The method is neither limited to large or high-throughput or any scale, and can be refined based on the availability of test compounds or other variable features of the screening assay.
The term "vector" refers to any linear or circular DNA construct comprising one of the nucleic acid molecules of the application. The vector can refer to an expression cassette or any recombinant expression system for the purpose of expressing one of the nucleic acid molecules of the application in vitro or in vivo, constitutively or inducibly, in any cell, including mammalian cells. The vector can remain episomal or integrate into the host cell genome. The vector can have the ability to self-replicate or not (i.e. drive only transient expression in a cell). The term includes recombinant expression cassettes that contain only the minimum elements needed fortranscription of the recombinant nucleic acid. The vector of the invention can a "recombinant vector" which is by definition a man-made vector. The vector can also be a viral vector including lentiviral, retroviral, adenoviral and adeno-associated viral vectors.
"Neuron" or "neurons" or "neuronal cells" as used herein are a type of cell in the central nervous system, which receive, integrate, and pass along information by releasing neurotransmitters. Said neurotransmitters are chemicals that cross-over from the terminal button at the end of an axon over the synapse to the neighbouring neuron. Non-limiting examples of neuron cells are primary cortical neurons, primary basal forebrain cholinergic neurons, primary neural stem cells, sensory neurons (e.g. retinal cells, olfactory epithelium cells), motor neurons (e.g. spinal motor neurons, pyramidal neurons, Purkinje cells) and interneurons (e.g. dorsal root ganglia cells).
The "action potential" as used herein is a rapid and reversible reversal of the electrical potential difference across the plasma membrane of excitable cells such as neurons, muscle cells and some endocrine cells. In a neuronal action potential, the membrane potential rapidly changes from its resting level of approximately -70 mV to around +50 mV and, subsequently, rapidly returns to the resting level again. The neuronal action potential forms an important basis for information processing, propagation and transmission. In muscle cells, the action potential precedes, and is necessary to bring about, muscle contraction. Some endocrine cells also exhibit action potentials, where the excitation leads to hormone secretion.
"Treatment" refers to any rate of reduction or retardation of the progress of the disease or disorder compared to the progress or expected progress of the disease or disorder when left untreated. More desirable, the treatment results in no or zero progress of the disease or disorder (i.e. "inhibition" or "inhibition of progression") or even in any rate of regression of the already developed disease or disorder. "Reduction" or "reducing" as used herein refers to a statistically significant reduction, more particularly said statistically significant reduction is an at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% reduction compared to the control situation.
"Increasing" or "increase" or "enhancing" or "promoting" or "stimulating" as used herein interchangeable and refer to a statistically significant increase, more particularly said statistically significant increase is an at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% increase compared to the control situation.
The term "statistically significantly" different is well known by the person skilled in the art. Statistical significance plays a pivotal role in statistical hypothesis testing. It is used to determine whether the null hypothesis should be rejected or retained. The null hypothesis is the default assumption that nothing happened or changed, hence that there is no difference for example in the activity of the NAVI.6 channel in the presence of a test compound compared to the activity of the NAVI.6 channel in the absence of said test compound. For the null hypothesis to be rejected, an observed result has to be statistically significant, i.e. the observed p-value is less than the pre-specified significance level a. The p-value of a result, p, is the probability of obtaining a result at least as extreme, given that the null hypothesis were true. In one embodiment, a is 0.05. In a more particular embodiment, a is 0.01. In an even more particular embodiment, a is 0.001.
By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an isolated FGF13A_S peptide as disclosed herein refers to a FGF13A derived peptide, which has been purified from the molecules which flank it in a naturally- occurring state, e.g. the C-terminal part of FGF13A has been removed. An isolated peptide can be generated by amino acid chemical synthesis or can be generated by recombinant production. Another example concerns an isolated neuronal cell, which refers to a neuronal cell which has been extracted and purified from the naturally-occurring state, involving tissue. An isolated neuronal cell preparation can be obtained from several neuronal tissue types using for example specialized commercial kits that make use of proteases to digest intercellular protein junctions followed by gentle mechanical disruption to liberate individual cells, or for instance but not limited to the exemplified method.
By "LRRC37B-FGF13A-SCN1B complex" as mentioned herein, it is meant that LRRC37B, FGF13A and SCN1B can be co-immunoprecipitated together, for example by transfection in HEK cells and from human tissue. By referring to a "complex", it is meant that components of said complex can be coimmunocoprecipitated together, for example, NAVa subunits, LRRC37B, FGF13A and SCN1B can be co- immunoprecipitated together (human cortical biopsy), LRRC37B, FGF13A and NAVI.6 can be co- immunoprecipitated together (transfection in HEK cells). By FGF13A S-fragment or S-domain, also referred to as "ExonS", it is meant a fragment consisting the 63 first amino acids of FGF13A protein encoded by its first specific alternative exon, said FGF13A S-fragment sequence being depicted in SEQ ID No. 4. By homologue of FGF13A S-fragment it is meant a protein or a peptide comprising at least 80% 81% 82% 83% 84% 85% 86°% 87% 88% 89% 90% 91% 92% 93% 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the FGF13A protein fragment depicted in SEQ ID No. 4, wherein the homologue has the same or the similar function or effect as the GFG13A protein fragment as depicted in SEQ ID No. 4.
By FGF13A protein fragment also referred to as "FGF13A fragment", as used herein, it is meant any protein or peptide fragment having at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% amino acid sequence identity with the FGF13A protein as depicted in SEQ ID No. 2. By homologue of said FGF13A fragment it is meant a protein or a peptide comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with said FGF13A fragment, wherein the homologue has the same or the similar function or effect as the GFG13A fragment.
A hominid-specific modulation of neuronal excitability
The inventors of current application have demonstrated that LRRC37B is an axon initial segment (AIS) hominid-specific protein sufficient to modulate neuronal excitability. The AIS is a unique subcellular compartment at the beginning of the axon. At the molecular level, AIS proteins organize as a layered scaffold spanning from microtubules to the plasma membrane (Leterrier 2016 Curr Top Membr 77: 185- 233). The central component of this scaffold is ankyrin G (ANK3) that concentrates at the AIS and anchors AIS-specific membrane proteins, including voltage-gated sodium (NAV) and potassium (Kv) channels.
Voltage-gated sodium channels (VGSCs) primarily function to provide an explosive, regenerative inward current during the rising phase of the action potential (AP) (Clark et al 2009 Neuroscientist 15:651-668; Kole and Stuart 2012 Neuron 73:235-247). At normal resting potentials, VGSCs will be in a closed confirmation and be non-conducting. VGSCs generally adopt an open conformation upon membrane depolarization (about -40mV) to allow a flow of sodium ions down a concentration gradient from the outside of the cell to the inside of the cell. In the brain, VGSCs are formed by an a subunit which constitutes the channel itself and beta subunits (SCN1-2-3-4B) which modulate its gate (Catterall et al 2005 Pharmacol Rev 57: 411-25). The a subunits (NAVa subunits) are further classified into nine subtypes (NAVI.1-1.9), the expression of which is both cell- and tissue-specific (Catterall et al 2005 Pharmacol Rev 57: 411-25). The main subtypes expressed in the brain are the NAVI.6 (coded by SCN8A) localized in the axonal initial segment, nodes of Ranvier, cell somas and dendrites; NAV1.1 is found in the cell soma; and NAVI.2 is present in unmyelinated axons and myelinated axons during development (Mantegazza et al 2010 Lancet Neurol 9: 413-424). It is generally accepted that abnormal expression of VGSCs contribute to the changes in intrinsic neuronal excitability that underlie several neurological disorders including epilepsy and migraine (Mantegazza et al 2010 Lancet Neurol 9: 413-424). For example, changes in mRNA and protein levels of NAV1.1, NAVI.2, NAVI.3, NAVI.6, as well as beta subunits have been shown in animal models of epilepsy and in human brain tissue in acquired epilepsy (Aronica et al 2001 Eur J Neurosci 13: 1261-1266; Blumenfeld et al 2009 Epilepsia 50: 44-55; Ketelaars et al 2001 Neuroscience 105: 109-120; Klein et al 2004 Brain Res 1000: 102-109). The most compelling evidence for the role VGSCs in epileptogenesis, however, comes from the observation that mutations in several sodium channel genes lead to inherited epileptic syndromes ranging in severity from mild forms such as febrile seizures (Mantegazza et al 2005 Proc Natl Acad Sci USA 102: 18177-18182) through to severe disorders including Dravet's syndrome (Claes et al 2001 Am J Hum Genet 68: 1327-1332; Dravet et al 2005 Adv Neurol 95: 71-102).
Current application teaches that the LRRC37B-mediated modulation of neuronal excitability comprises several interacting proteins. This is schematically illustrated in Figure 7. First, it is herein demonstrated that LRRC37B interacts with FGF13A that on its turn binds to NAVI.6, the VGSC that is most prominent at the AIS and that is known to generate APs (Inda et al 2006 Proc Natl Acad Sci U S A 103: 2920-2925; Van Wart et al 2007 J Comp Neurol 500: 339-352; Lorincz and Nusser 2008 J Neurosci 28: 14329-14340; Dumenieu et al 2017 Front Cell Neurosci 11:115). In particular, LRRC37B concentrates inhibitory effects of FGF13A on the channel function of voltage-gated sodium channel, thereby reducing neuronal excitability, specifically at the AIS level. The binding between FGF13A and NAVI.6 inhibits NAVI.6 action. "NAVI.6" and "NAVI.6 voltage-gated sodium channels" are used as synonyms throughout the description of the present invention. Interestingly, LRRC37B or FGF13A overexpression as well as extracellular administration of the FGF13A fragment binding to LRRC37B decreases neuron excitability.
Second, it is herein demonstrated that LRRC37B binds SCN1B and that the LRRC37B fragment that binds SCN1B is sufficient to abolish the SCN1B-NAV1.6 molecular interaction. SCN1B is a NAV p-subunit that modulates VGSCs, in particular NAVI.6 (Wimmer et al 2010 J Clin Invest 120: 2661-2671).
The NAV -subunit family consists of four proteins: pi-4, coded by genes SCN1B to SCN4B, respectively. These are single-span transmembrane proteins oriented with the amino terminus facing the extracellular space. The extracellular domain presents a conserved immunoglobulin domain, homologous to the one in cell adhesion molecules. The carboxyl terminus associates with cytoskeletal and scaffolding proteins, pi (or SCN1B) and P2 associate with ankyrin-G and ankyrin-B in both brain and heart, and their interaction is critical for channel surface expression and modulates the channel function in vivo (Cerrone et al 2014 Cardiac Electrophysiology). P-subunits function in concert with a-subunits to promote channel trafficking to the plasma membrane and to modulate the NAV biophysical properties (Deschenes 2018 Cardiac Electrophysiology). While there are some discrepancies in some of the observations, one consensus is with the increase in current density seen when expressing SCN1B with the different NAVa isoforms. Coexpression of SCN1B subunits with NAVI.5 in vitro leads to increased a-subunit expression at the plasma membrane resulting in an augmentation in current density (Nuss et al 1995 J Gen Physiol 106: 1171-1191; Qu et al 1995 J Biol Chem 270: 25696-25701; Isom et al 1995 J Biol Chem 270: 3306-3312).
The finding that LRRC37B - besides recruiting FGF13A to the AIS and therefore increasing the latter's inhibitory activity on NAVI.6 which is involved in epilepsy - also binds SCN1B is of particular interest because it was previously reported that mutations in the SCN1B gene cause human epilepsy (Wallace et al 1998 Nat Genet 19: 366-370; Scheffer et al 2007 Brain 130: 100-109; Patino et al 2009 J Neurosci 29: 10764-10778). In vitro studies of the human epilepsy mutation SCN1B(C121W) suggest a disease mechanism caused by loss of modulatory function (Meadows et al 2002 J Neurosci 22: 10699-10709; Xu et al 2007 NeuroScience 148: 164-174).
The data reported in the Examples are consistent with a model whereby FGF13A, secreted by pyramidal neurons or other cortical cell types, can bind to and block the function of NAVI.6, resulting in decreased excitability. LRRC37B enhances this effect by concentrating FGF13A at the level of the AIS, resulting in a further decrease in excitability. Additionally, LRRC37B also modulates neuronal excitability by blocking the interactions between SCN1B and NAVI.6 (Figure 7).
Intriguingly, mouse neurons that obviously lack the herein disclosed hominid-specific regulation of neuron excitability are less excitable compared to human neurons (Beaulieu-Laroche et al 2018 Cell 175: 643-651. el4; Beaulieu-Laroche et al 2021 Physiol Rev 99: 1079-1151). This makes the LRRC37B- dependent regulation of particular interest to understand the molecular basis of human neuronal evolution. Electrophysiological recordings in human versus other mammalian species like rodents (mouse, rat) or non-human primates (macaque, marmoset) recently showed that human pyramidal neurons display enhanced signal processing abilities, but also a decreased excitability (Beaulieu-Laroche et al 2018 Cell 175: 643-651. el4; Beaulieu-Laroche et al 2021 Physiol Rev 99: 1079-1151; Deitcher et al 2017 Cereb Cortex 27: 5398-5414). The mechanisms underlying these differences have been linked so far to specific properties of the dendrites. This disclosure is the first report demonstrating the participation of the AIS, more particularly of a LRRC37B-mediated output modulation. Therapeutic applications
LRRC37B can affect information processing through neuronal gain modulation, by which neurons adapt to changing inputs (Ferguson and Cardin 2020 Nat Rev Neurosci 21: 80-92). Neuronal gain modulation relies largely on the regulation of synaptic inputs, but also output modulation, including at the level of the AIS (Debanne et al 2019 Curr Opin Neurobiol 54: 73-82). Moreover, neuronal plasticity and learning involves modulation of the neuronal gain including at the level of the output (Ferguson and Cardin 2020 Nat Rev Neurosci 21: 80-92; Jamann et al 2021 Nat Commun 12: 1-14.). The modulation of neuronal excitability by LRRC37B could thus influence sensory processing and plasticity, at least in part through gain modulation, and thereby contribute to human-specific properties of cortical circuits.
The discovery of LRRC37B and its binding partners has also interesting implications for brain diseases, in particular epilepsy. Indeed, LRRC37B binds to FGF13A and to SCN1B, which are both mutated in severe forms of epilepsy (Devinsky et al 2018 Epilepsy Nat Rev Dis Prim 4: 18024; Wimmer et al 2010 Physiol 588: 1829-1840). Hence, besides the particular interest of the LRRC37B-FGF13A-SCN1B complex and its modulation of NAVI.6 in the treatment of these epilepsy patients, FGF13A peptide administration and/or administration of the B peptide encoding the B-specific domain of LRRC37B is of high relevance in modulating neuronal excitability and for treating epilepsy and seizures in general.
Therefore, in a first aspect, a modulator of the interaction between a LRRC37B protein and a NAVI.6 voltage-gated sodium channel, in particular a modulator of the LRRC37B-FGF13A-SCN1B, the LRRC37B- FGF13A, the FGF13A-NAV1.6, the SCN1B-NAV1.6, the LRRC37B-SCN1B or the LRRC37B-FGF13A-SCN1B- NAV1.6 interactions is provided for use as a medicament. In a preferred embodiment, a modulator of the LRRC37B protein and NAVI.6 voltage-gated sodium channel functions for use as a medicine is provided.
This is equivalent as saying that a method of treatment is provided comprising the step of administering to a subject a modulator of the LRRC37B-FGF13A-SCN1B, LRRC37B-FGF13A, FGF13A-NAV1.6, SCN1B- NAV1.6, LRRC37B-SCN1B and/or the LRRC37B-FGF13A-SCN1B-NAV1.6 interactions. In a particular embodiment, the modulator is provided for modulating neuron excitability and/or function. Several neurological disorders are known that are caused by an aberrant modulation of neuron excitability. Nonlimiting examples are seizures, epilepsy, epileptogenesis, neuropathic pain, anxiety, depression, Alzheimer's disease, cognitive impairments, dystonia, narcolepsy and spasticity. In a particular embodiment, the modulator of the LRRC37B-FGF13A-SCN1B, LRRC37B-FGF13A, FGF13A-NAV1.6, SCN1B-NAV1.6, LRRC37B-SCN1B and/or the LRRC37B-FGF13A-SCN1B-NAV1.6 interactions is provided for use to treat seizures, epilepsy or epileptogenesis. "Seizures" as used herein refers to paroxysmal alteration of neurologic function caused by the excessive, hypersynchronous discharge of neurons in the brain (Stafstrom & Carmant 2015 Cold Spring Harb Perspect Med 5(6): a022426). They can be generalized (leading to absences, tonic-clonic, myoclonic or atonic manifestations) or focal (manifestations are linked to the brain area affected) (Stafstrom & Carmant 2015 Cold Spring Harb Perspect Med 5(6): a022426). Based on their etiology, seizures are also classified into symptomatic (lesion, syndrome) or idiopathic seizures.
"Epilepsy" is a chronic non-communicable disease of the brain that affects around 50 million people worldwide. It is characterized by recurrent seizures.
"Epileptogenesis" as used herein is the process whereby a previously normal brain is functionally altered and biased towards the generation of the abnormal paroxysmal electrical activity that defines chronic seizures.
In one embodiment, the modulator statistically significantly improves, stimulates or enhances the interaction between LRRC37B-FGF13A-SCN1B, LRRC37B-FGF13A, FGF13A-NAV1.6, SCN1B-NAV1.6, LRRC37B-SCN1B and/or the LRRC37B-FGF13A-SCN1B-NAV1.6 compared to a situation where the modulator is absent. Hence, the modulator is an agonist of the LRRC37B-FGF13A-SCN1B, LRRC37B- FGF13A, FGF13A-NAV1.6, SCN1B-NAV1.6, LRRC37B-SCN1B and/or the LRRC37B-FGF13A-SCN1B-NAV1.6. An enhanced interaction of LRRC37B and FGF13A or of FGF13A and NAVI.6, inhibits the NAVI.6 output and thus leads to a decreased neuron excitability. An enhanced interaction of LRRC37B and SCN1B reduces the binding between SCN1B and NAVI.6 and thus lead to a decreased neuron excitability.
In one embodiment, the modulator statistically significantly inhibits, decreases, reduces or blocks the interaction between LRRC37B-FGF13A, FGF13A-NAV1.6 and/or LRRC37B-SCN1B compared to a situation where the modulator is absent. Hence, the modulator is an antagonist or inhibitor of the LRRC37B- FGF13A interaction, the FGF13A-NAV1.6 interaction or of the LRCC37B-SCN1B interaction. Such reduced interaction overcomes FGF13A inhibition of NAVI.6 and thus lead to an increase neuron excitability, while a reduced interaction between LRRC37B and SCN1B leads to a reduced competition between SCN1B and NAVI.6 and thus to an increased neuron excitability.
In a preferred embodiment, said modulator of the interaction between the LRRC37B protein and the NAVI.6 voltage-gated sodium channel for use as a medicine, is a protein or peptide, said protein or peptide comprising FGF13A or a FGF13A fragment or a peptidomimetic thereof. Preferably said modulator is a peptide comprising a FGF13A fragment, said FGF13A fragment being an S- domain of FGF13A or FGF13A S-fragment or ExonS or a peptidomimetic thereof. In a particularly preferred embodiment, said FGF13A fragment comprises a peptide of SEQ. ID No.15 or SEQ ID No. 4 or a peptidomimetic thereof. In a further preferred embodiment, FGF13A fragment comprises a peptide of SEQ ID No.15. In a further preferred embodiment, said FGF13A fragment consists of SEQ ID No.15 or SEQ ID No. 4 or a peptidomimetic thereof.
Modulating the LRRC37B-FGF13A interaction
It is demonstrated herein that the hominid-specific LRRC37B forms a complex with FGF13A and NAVI.6 at the AIS of cortical neurons. Interestingly, overexpressing LRRC37B or FGF13A or extracellularly administering FGF13A or the S-fragment of FGF13A decreases excitability of cortical neurons.
Therefore, in a second aspect the application provides a modulator of neuron excitability, wherein the modulator is the FGF13A protein or a peptidomimetic thereof, preferably a FGF13A protein fragment comprising or consisting of the S-fragment, referred herein to as a S-domain or ExonS, or a peptidomimetic thereof, the LRRC37B protein or a peptidomimetic thereof, the LRRC37B protein fragment comprising the B specific domain or a peptidomimetic thereof or any of the FGF13A, FGF13A S-fragments or LRRC37B proteins or protein fragments described herein. It should be understood that the S-fragment and S-domain of the FGF13A protein or ExonS, can be used as synonyms throughout the description.
FGF13A as used herein refers to the Fibroblast Growth Factor 13A (Uniprot ID: Q92913-1) and is also known as FHF2A (Fibroblast growth factor homologous factor). Its cDNA sequence is depicted in SEQ ID No. 1, while its amino acid sequence is depicted in SEQ ID No. 2.
SEQ ID No. 1 (cDNA FGF13A)
ATGGCGGCGGCTATCGCCAGCTCGCTCATCCGTCAGAAGAGGCAAGCCCGCGAGCGCGAGAAATCCAACGCCT GCAAGTGTGTCAGCAGCCCCAGCAAAGGCAAGACCAGCTGCGACAAAAACAAGTTAAATGTCTTTTCCCGGGTC AAACTCTTCGGCTCCAAGAAGAGGCGCAGAAGAAGACCAGAGCCTCAGCTTAAGGGTATAGTTACCAAGCTATA CAGCCGACAAGGCTACCACTTGCAGCTGCAGGCGGATGGAACCATTGATGGCACCAAAGATGAGGACAGCACTT ACACTCTGTTTAACCTCATCCCTGTGGGTCTGCGAGTGGTGGCTATCCAAGGAGTTCAAACCAAGCTGTACTTGG CAATGAACAGTGAGGGATACTTGTACACCTCGGAACTTTTCACACCTGAGTGCAAATTCAAAGAATCAGTGTTTG AAAATTATTATGTGACATATTCATCAATGATATACCGTCAGCAGCAGTCAGGCCGAGGGTGGTATCTGGGTCTGA ACAAAGAAGGAGAGATCATGAAAGGCAACCATGTGAAGAAGAACAAGCCTGCAGCTCATTTTCTGCCTAAACCA CTGAAAGTGGCCATGTACAAGGAGCCATCACTGCACGATCTCACGGAGTTCTCCCGATCTGGAAGCGGGACCCC AACCAAGAGCAGAAGTGTCTCTGGCGTGCTGAACGGAGGCAAATCCATGAGCCACAATGAATCAACGTAG SEQ ID No. 2 (amino acid sequence FGF13A)
MAAAIASSLIRQKRQAREREKSNACKCVSSPSKGKTSCDKNKLNVFSRVKLFGSKKRRRRRPEPQLKGIVTKLYSRQGY HLQLQADGTIDGTKDEDSTYTLFNLIPVGLRVVAIQGVQTKLYLAMNSEGYLYTSELFTPECKFKESVFENYYVTYSSMI YRQQQSGRGWYLGLNKEGEIMKGNHVKKNKPAAHFLPKPLKVAMYKEPSLHDLTEFSRSGSGTPTKSRSVSGVLNG GKSMSHNEST
In one embodiment, the application provides the FGF13A protein as depicted in SEQ ID No. 2 or a homologue thereof. A "homologue thereof" as used in current application refers to a protein or protein fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity compared to the reference protein (e.g. FGF13A protein as depicted in SEQ ID No. 2). Importantly, the homologue should comprise the same or similar effect as the reference protein (e.g. FGF13A protein as depicted in SEQ ID No. 2). However, it is important to highlight herein that modifications to the amino acid sequences are still within the scope of the application as long as the homologue still has the same or similar effect (e.g. reducing NAVI.6 activity and therefore neuron excitability). These modifications comprise those known to the skilled person and include, for example, the substitution of one or more hydrophobic amino acids, preferably surface-exposed hydrophobic amino acids, with one or more hydrophilic amino acids. In one embodiment, the modifications comprise the substitution of up to 10, 9, 8, 7, 6, 5, 4, 3 or 2, hydrophobic amino acids, preferably surface-exposed hydrophobic amino acids, with hydrophilic amino acids. In a particular embodiment, a homologue consists of an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to amino acid sequence of the reference protein. Hence, an FGF13A homologue is provided consisting of an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to SEQ ID No. 2. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.
"Conservative substitutions" may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As used herein, "conservative substitutions" are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt a-helices.
As used herein, "non-conservative substitutions" are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above. In various embodiments, the substitutions may also include non-classical amino acids (e.g. selenocysteine, pyrrolysine, N-formylmethionine p-alanine, GABA and 6-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4- aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, p- alanine, fluoro-amino acids, designer amino acids such as p methyl amino acids, C a-methyl amino acids, N a-methyl amino acids, and amino acid analogs in general).
Also the nucleic acid sequence encoding the FGF13A protein as depicted in SEQ ID No. 2 or any of the homologues thereof described above is provided herein, more particularly the nucleic acid sequence as depicted in SEQ ID No. 1. A host cell is provided comprising the nucleic acid sequence, more particularly heterologously or recombinantly expressing the nucleic acid sequence. A non-limiting example of a host cell is a HEK cell.
In another embodiment, the application provides a FGF13A peptide or protein fragment of at most 100 amino acids, at most 90, at most 80 or at most 70 amino acids comprising the amino acid sequence as depicted in SEQ ID No. 4. SEQ ID No. 4 depicts the 63 amino acids long sequence encoded by the S exon of the FGF13A gene, referred to herein as the FGF13A_S peptide or as FGF13A S-fragment or as the FGF13A S-domain or as ExonS.
SEQ ID No. 3 (cDNA FGF13A_S)
ATGGCGGCGGCTATCGCCAGCTCGCTCATCCGTCAGAAGAGGCAAGCCCGCGAGCGCGAGAAATCCAACGCCT GCAAGTGTGTCAGCAGCCCCAGCAAAGGCAAGACCAGCTGCGACAAAAACAAGTTAAATGTCTTTTCCCGGGTC AAACTCTTCGGCTCCAAGAAGAGGCGCAGAAGAAGACCAGAG
SEQ ID No. 4 (amino acid sequence FGF13A_S)
MAAAIASSLIRQKRQAREREKSNACKCVSSPSKGKTSCDKNKLNVFSRVKLFGSKKRRRRRPE
In a particular embodiment, the application provides the FGF13A protein fragment consisting of SEQ ID No. 4. Said FGF13A protein fragment consisting of SEQ ID No. 4 is referred to as an FGF13A S-fragment or an ExonS in the present application. Also a homologue is provided of the FGF13A S-fragment according to the definition above, hence comprising at least 90% amino acid identity with the FGF13A protein fragment as depicted in SEQ ID No. 4, wherein the homologue has the same or similar function or effect as the FGF13A protein S-fragment as depicted in SEQ ID No. 4. In another embodiment, an FGF13A protein fragment is provided consisting of an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to SEQ ID No. 4, wherein the FGF13A protein fragment still has the same or similar activity compared to SEQ ID No. 4. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.
In a preferred embodiment, a further fragment of FGF13A S-fragment (i.e. ExonS) is provided. In a particularly preferred embodiment, said fragment comprises a sequence as depicted in SEQ ID No. 15 or a homologue thereof. By homologue it is meant a peptide which differs in ten amino acids, six amino acids, five amino acids, four amino acids, three amino acids, preferably two amino acids, most preferably one amino acid to the SEQ ID No. 15. In one embodiment, a further fragment of said FGF13A S-fragment or ExonS is provided consisting of an amino acid sequence having one, or two, three, four, five, six or ten amino acid mutations with respect to SEQ ID No. 15. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions. Said fragment depicted as SEQ ID 15 consists of 24 amino acid long sequence, and is referred herein to as "ExonS 40-63".
In another embodiment, a fragment of FGF13A S-fragment is provided, said fragment comprising SEQ ID No. 16 or a homologue thereof, preferably consisting of SEQ ID No. 16. By homologue it is meant a peptide which differs in ten amino acids, six amino acids, five amino acids, four amino acids, three amino acids, preferably two amino acids, most preferably one amino acid to the SEQ ID No. 16. In one embodiment, a further fragment of said FGF13A S-fragment or ExonS is provided consisting of an amino acid sequence having one, or two, three, four, five, six or ten amino acid mutations with respect to SEQ ID No. 16. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions. Said fragment depicted as SEQ ID 16 consists of 20 amino acid long sequence referred herein to as "ExonS 4-23". SEQ ID No. 15 (amino acid sequence FGF13A_S 40-63)
KNKLNVFSRVKLFGSKKRRRRRPE
SEQ ID No. 16 (amino acid sequence FGF13A_S 4-23)
AIASSLIRQKRQAREREKSN
SEQ ID No. 17 (cDNA ExonS 40-63)
AAAAACAAGTTAAATGTCTTTTCCCGGGTCAAACTCTTCGGCTCCAAGAAGAGGCGCAGAAGAAGACCAGAG
SEQ ID No. 18 (cDNA ExonS 4-23)
GCTATCGCCAGCTCGCTCATCCGTCAGAAGAGGCAAGCCCGCGAGCGCGAGAAATCCAAC
In one embodiment, the amino acid sequence depicted in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 15 or SEQ ID No. 16 or the homologue thereof refers to an isolated protein or peptide. According to another embodiment, the amino acid sequence depicted in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 15 or SEQ ID No. 16 or the homologue thereof is generated by chemical amino acid synthesis. According to another embodiment SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 15 or SEQ ID No. 16 or the homologue thereof is generated by recombinant production. It should be understood that a peptide of a sequence SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 15 or SEQ ID No. 16 could be provided by any suitable method known to a person skilled in the art, without departing from the scope of the present invention.
Also provided is a molecule comprising the FGF13A protein as depicted in SEQ ID No. 2, or FGF13A fragment SEQ ID No. 4, SEQ ID No. 15 or SEQ ID No. 16, or a homologue thereof and an additional entity. Said additional entity can be a biological, chemical or another protein or protein fragment. In one embodiment, said entity is a half-life extension entity and/or an entity that facilitates the molecule to cross the blood brain barrier. In particular embodiments, said molecule is a chimeric molecule, a chimeric protein, a dimeric protein, a fusion protein, a composition, a combination, a peptide or a polypeptide.
Also provided is a pharmaceutical composition comprising an FGF13A protein fragment, wherein the FGF13A protein fragment is the FGF13A S-fragment as depicted in SEQ ID No. 4, or a FGF13A fragment comprising SEQ ID No.15 or SEQ ID No. 16 or any of the homologues thereof as described herein. In a particularly preferred embodiment, the pharmaceutical composition comprising the FGF13A fragment, said FGF13A fragment comprising SEQ ID NO. 15, preferably consisting of SEQ ID NO. 15 or a homologue thereof is provided.
In a particular embodiment, a peptidomimetic of the FGF13A S-fragment or a peptidomimetic of a FGF13A fragment comprising SEQ ID No.15 or SEQ ID No. 16, is provided. "Peptidomimetic" as used herein refers to a non-natural peptide or peptide comprising at least one non-natural amino acid. Peptidomimetics provide an alternative source of potent and selective Protein-Protein Interaction (PPI) modulators and occupy the chemical gap between small molecules and biologies, such as antibodies. In one embodiment, the FGF13A S-fragment or peptidomimetic thereof comprises at least one D-amino acid. All amino acids (except for glycine) have two different stereoisomers or mirror images of their structure. These are labelled L (left-handed) and D (right-handed) to distinguish the mirror images. L- amino acids occur in all proteins produced by animals, plants, fungi and bacteria. In the Fisher projection, the amine group of L-amino acids occurs on the left side. In contrast, in D-amino acids, the amine group occurs in the right side in the Fisher projection. D-Amino acids are only occasionally found in nature as residues in proteins. The amino acids that make up the proteins in mammals are all L-amino acids. Hence, naturally occurring human proteins or peptides do not comprise D-amino acids.
Besides natural amino acids, non-natural or unnatural amino acids have been developed. Non-natural amino acids are so called because they are not found in natural polypeptide chains. They are not among the 20 amino acids attached to tRNAs in living cells used to polymerize proteins. Some unnatural amino acids do occur naturally, but most are chemically synthesized. They can for example be made through chemical modifications of natural amino acids, such as N-methyl amino acids (attachment of a methyl group to the nitrogen in the amino group), a-methyl amino acids (a methyl group replaces the hydrogen on the a carbon), beta-amino acids (addition of a second carbon between the amino group and carboxy groups), homo-amino acids (addition of a methylene group between the a carbon and the side group) or beta-homo-amino acids (addition of a second carbon between the amino and carboxy groups and the addition of a methylene group between the a carbon and the side group). Unnatural amino acids are valuable building blocks in the manufacture of a wide range of pharmaceuticals. Non-natural amino acids can exhibit biological activity as free acids and they can be incorporated into linear or cyclic peptides with biological activity. In one embodiment, the FGF13A S-fragment or the FGF13A fragment or peptidomimetic thereof comprises at least one non-natural amino acid. Non-limiting examples of non- natural amino acids are isoethylmethyl-benzene, 6-chloro-3-methyl-lH-indole, methylcyclohexane, ethylcyclohexane, 2-naphthalene, ethylbenzene, l,l-difluoro-4-cyclohexyl, 4-methyl-l-methoxy-2- methylbenzene, l-chloro-4-methylbenzene, 4-methylphenyl-methanol, 3-methylbenzoic acid and 4- methylaniline.
The application also provides a nucleic acid molecule encoding the FGF13A S-fragment or FGF13A fragment or homologues thereof as described above, more particularly a nucleic acid molecule as depicted in SEQ. ID No. 3 or SEQ ID No. 17, respectively. In another embodiment, a vector is provided comprising the nucleic acid molecule encoding the FGF13A protein as depicted in SEQ ID No. 2 or the FGF13A fragments as depicted in SEQ ID No. 4 or SEQ ID No. 15.
Also a host cell is provided comprising the nucleic acid molecule or the vector, more particularly heterologously expressing the nucleic acid molecule or with recombinant protein application. A nonlimiting example of a host cell is a HEK cell.
In another independent aspect, the present application concerns an isolated FGF13A protein fragment, preferably an isolated FGF13A S fragment, more preferably a peptide comprising SEQ ID No. 15, a peptidomimetic thereof or a nucleic acid molecule encoding said FGF13A protein fragment, FGF13A S- fragment as depicted in SEQ ID No. 4 or the peptide comprising SEQ ID No. 15 for use as a medicine. Said FGF13A protein fragment, FGF13A S-fragment and/or the peptide comprising SEQ ID No. 15 were found to be particularly efficient in modulating the neuronal excitability of human cortical pyramidal neurons. In a preferred embodiment, FGF13A, the FGF13A protein fragment, the FGF13A S fragment and/or the peptide comprising SEQ ID No. 15 or SEQ ID No. 4 are used to decrease neuron excitability, even more particularly for use to treat seizures, epilepsy, epileptogenesis, ataxia, autism spectrum disorder, intellectual disability, cardiac arrythmia or pain.
As demonstrated in the Example section, overexpression of LRRC37B reduces the neuronal excitability. LRRC37B acts at least in part through FGF13A on the NAVI.6 sodium channel. The interaction between FGF13A and LRRC37B is established through binding of the FGF13A S-fragment and LRR domain of LRRC37B respectively. The LRR domain of LRRC37B is depicted in SEQ ID No. 8.
In one embodiment, the application provides the LRRC37B protein as depicted in SEQ ID No. 6 or a homologue thereof, more particular a homologue of at least 90% identity over the full length of LRRC37B, wherein the homologue has the same or similar function as the LRRC37B domain as depicted in SEQ ID No. 6. In another embodiment, an LRRC37B protein is provided consisting of an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to SEQ ID No. 6, wherein the protein still has the same or similar activity compared to SEQ ID No. 6. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.
In another embodiment, a nucleic acid sequence is provided encoding the LRRC37B or homologue thereof, particularly encoding the amino acid sequence as depicted in SEQ ID No. 6, more particularly a nucleic acid sequence as depicted in SEQ. ID No. 5. Also a host cell is provided comprising the nucleic acid sequence, more particularly heterologously expressing the nucleic acid sequence or with recombinant protein application. A non-limiting example of a host cell is a HEK cell.
SEQ ID No. 5 (cDNA LRRC37B)
ATGTCTTGGCTGCGTTTCTGGGGCCCATGGCCCCTCCTTACGTGGCAACTATTGTCTTTACTAGTCAAGGAGGCTC
AGCCTCTGGTGTGGGTCAAGGACCCGCTCCAGCTGACCTCTAACCCCCTGGGGCCACCTGAGCCCTGGTCTTCCC
GCTCCTCCCATCTCCCATGGGAATCTCCCCATGCACCTGCTCCCCCAGCAGCCCCGGGGGACTTTGATTACCTGGG GCCCTCTGCTTCTTCGCAGATGTCAGCCCTGCCTCAGGAACCAACTGAAAATTTGGCTCCATTCCTGAAGGAATTG GATTCAGCTGGAGAGCTGCCCCTGGGGCCAGAGCCGTTCTTGGCTGCACATCAGGACTTAAATGACAAGCGGAC TCCAGAAGAAAGGCTCCCAGAGGTGGTTCCGCTTCTCAACCGGGATCAGAACCAGGCCCTAGTTCAGCTTCCTCG CCTCAAGTGGGTTCAAACTACAGATCTAGATCGGGCTGCAGGTCATCAGGCAGATGAAATACTTGTTCCACTAGA CAGTAAGGTTTCAAGACCAACCAAATTTGTTGTTTCGCCCAAGAACCTGAAGAAAGATCTAGCTGAACGTTGGAG CCTTCCTGAGATTGTTGGGATTCCACACCAATTATCCAAACCTCAGCGTCAGAAACAGACTTTGCCAGATGATTAT TTGAGTATGGACACACTGTATCCCGGCAGCCTACCTCCAGAACTCCGGGTGAACGCAGATGAGCCTCCAGGGCC TCCTGAGCAAGTTGGACTTTCTCAATTCCATCTAGAGCCCAAAAGTCAAAATCCAGAGACCCTTGAAGACATCCA GTCCTCTTCACTCCAGGAAGAAGCCCCAGCGCAGCTTCTACAGCTCCCTCAGGAGGTAGAACCTTCAACCCAGCA GGAGGCCCCAGCTCTGCCTCCAGAGTCCTCTATGGAGAGTCTAGCTCAAACTCCACTGAATCATGAAGTGACAGT TCAACCTCCAGGTGAGGATCAAGCTCATTATAATTTGCCCAAGTTTACAGTCAAACCTGCAGATGTGGAGGTTAC CATGACTTCAGAGCCTAAAAATGAGACAGAATCTACCCAAGCCCAGCAGGAGGCCCCAATTCAGCCTCCCGAGG AGGCGGAACCTTCTTCTACAGCCCTGAGGACTACAGATCCTCCTCCAGAACACCCTGAGGTGACACTTCCACCTTC AGACAAGGGTCAGGCTCAGCATTCACACCTGACTGAAGCCACAGTTCAACCTCTGGACCTGGAGCTTAGCATAA CTACAGAGCCTACTACAGAGGTTAAACCGTCTCCAACCACGGAGGAAACCTCAGCTCAGCCTCCAGACCCGGGG CTTGCCATAACTCCAGAACCCACTACAGAGATTGGACATTCCACAGCCCTGGAGAAGACTAGAGCTCCTCATCCA GACCAGGTTCAGACTCTGCATCGAAGCCTGACTGAAGTCACAGGTCCACCTACAAAGTTAGAATCTTCGCAGGAT TCATTGGTGCAGTCTGAAACTGCACCAGAGGAACAGAAGGCCTCCACAAGCACCAACATATGTGAGCTCTGCAC CTGCGGAGATGAGACTCTGTCATGTGTTGGTCTCAGCCCAAAGCAGAGGCTCCGCCAAGTGCCTGTGCCAGAGC CCGACACCTACAATGGCATCTTCACCACCTTAAATTTCCAAGGAAACTATATTTCATACCTTGATGGAAATGTATG GAAAGCATACAGTTGGACCGAGAAACTAATTCTCAGTGAAAATTATTTGACTGAATTACCTAAGGATTCATTTGA AGGCCTGCTATACCTCCAGTATTTAGATTTATCCTGCAATAAAATACGATATATTGAAAGACAAACATTTGAATCA CTACCATTTTTGCAGTATATAAATCTGGGCTGCAATTTAATTACAAAACTGAGCCTTGGAACATTTCAGGCCTGGC ACGGAATGCAGTTTTTACACAACTTAATTCTCAATCGCAATCCTCTGACTACTGTCGAAGATCCATATCTCTTTGAA CTGCCGGCATTAAAATATCTAGACATGGGAACAACACACATCACACTTACAACACTTAAGAACATTCTCACGATG ACTGTTGAACTGGAAAAACTGATCTT ACCT AGCCATATGGCCTGCTGCCTCTGCCAATTT AAA AATAGCATTGAG GCTGTCTGCAAGACAGTCAAGCTGCATTGCAACACTGCATGTCTGACTAACAGCATACATTGTCCTGAAGAAGCA TCTGTAGGGAATCCAGAAGGAGCGTTCATGAAGATGTTACAAGCCCGGAAGCAGCACATGAGCACTCAGCTGAC TATTGAGTCGGAGGCGCCCTCAGACAGCAGTGGCATCAACTTGTCAGGCTTTGGGGGTGATCAGCTTGAAATTC AGCTAACCGAGCAGCTACGGTCCCTCATCCCCAACGAGGATGTGAGAAAGTTCATGTCTCATGTTATCCGGACCT TGAAAATGGAATGTTCAGAAACACATGTGCAAGGGAGCTGTGCCAAGCTCATGTTGCGAACAGGCCTCCTGATG AAGCTTCTCAGCGAGCAGCAGGAAGCAAAGGCATTGAATGTAGAATGGGATACGGACCAACAAAAAACAAATT ATATTAATGAGAACATGGAACAGAATGAACAGAAAGAGCAGAAGTCAAGTGAGCTCATGAAAGAAGTTCCAGG AGATGACTATAAGAACAAACTCATCTTCGCAATATCTGTGACTGTAATACTAATAATTTTGATTATAATTTTTTGTC TTATAGAGGTGAATTCACATAAAAGGGCATCAGAAAAATACAAAGACAACCCATCAATATCAGGAGCC
SEQ ID No. 6 (amino acid sequence LRRC37B)
MSWLRFWGPWPLLTWQLLSLLVKEAQPLVWVKDPLQLTSNPLGPPEPWSSRSSHLPWESPHAPAPPAAPGDFDYL GPSASSQMSALPQEPTENLAPFLKELDSAGELPLGPEPFLAAHQDLNDKRTPEERLPEVVPLLNRDQNQALVQLPRLK WVQTTDLDRAAGHQADEILVPLDSKVSRPTKFVVSPKNLKKDLAERWSLPEIVGIPHQLSKPQRQKQTLPDDYLSMD TLYPGSLPPELRVNADEPPGPPEQVGLSQFHLEPKSQNPETLEDIQSSSLQEEAPAQLLQLPQEVEPSTQQEAPALPPES SMESLAQTPLNHEVTVQPPGEDQAHYNLPKFTVKPADVEVTMTSEPKNETESTQAQQEAPIQPPEEAEPSSTALRTT DPPPEHPEVTLPPSDKGQAQHSHLTEATVQPLDLELSITTEPTTEVKPSPTTEETSAQPPDPGLAITPEPTTEIGHSTALE KTRAPHPDQVQTLHRSLTEVTGPPTKLESSQDSLVQSETAPEEQKASTSTNICELCTCGDETLSCVGLSPKQRLRQVPV PEPDTYNGIFTTLNFQGNYISYLDGNVWKAYSWTEKLILSENYLTELPKDSFEGLLYLQYLDLSCNKIRYIERQTFESLPFL QYINLGCNLITKLSLGTFQAWHGMQFLHNLILNRNPLTTVEDPYLFELPALKYLDMGTTHITLTTLKNILTMTVELEKLIL PSHMACCLCQFKNSIEAVCKTVKLHCNTACLTNSIHCPEEASVGNPEGAFMKMLQARKQHMSTQLTIESEAPSDSSGI NLSGFGGDQLEIQLTEQLRSLIPNEDVRKFMSHVIRTLKMECSETHVQGSCAKLMLRTGLLMKLLSEQQEAKALNVE WDTDQQKTNYINENMEQNEQKEQKSSELMKEVPGDDYKNKLIFAISVTVILIILIIIFCLIEVNSHKRASEKYKDNPSISG A
In another embodiment, the application provides a LRRC37B protein fragment consisting of SEQ ID No. 8 or a homologue thereof, more particularly a homologue of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over the LRRC37B protein fragment's full length. SEQ ID No. 8 depicts the amino acid sequence of the LRR domain of the LRRC37B protein, referred to herein as the LRRC37B-LRR peptide. In yet another embodiment, an LRRC37B protein fragment is provided consisting of an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to SEQ ID No. 8, wherein the LRRC37B protein fragment still has the same or similar activity compared to SEQ ID No. 8. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions. Also provided is a molecule comprising the LRRC37B-LRR peptide as depicted in SEQ ID No. 8, or a homologue thereof and an additional entity. Said additional entity can be a biological, chemical or another protein or protein fragment. In one embodiment, said entity is a half-life extension entity and/or an entity that facilitates the molecule to cross the blood brain barrier. In particular embodiments, said molecule is a chimeric molecule, a chimeric protein, a dimeric protein, a fusion protein, a composition, a combination, a peptide or a polypeptide.
In a particular embodiment, a peptidomimetic of the LRRC37B-LRR peptide is provided comprising at least one non-natural amino acid and/or D-amino acid. Also provided is a pharmaceutical composition comprising the LRRC37B-LRR peptide as depicted in SEQ ID No. 8 or any of the homologues or peptidomimetics thereof as described herein.
The application also provides a nucleic acid molecule encoding the LRRC37B-LRR peptide or homologue thereof, particularly encoding the amino acid sequence as depicted in SEQ ID No. 8, more particularly a nucleic acid molecule as depicted in SEQ ID No. 7. Also provided is a vector comprising any of the nucleic acid molecules. A host cell is also provided comprising the nucleic acid molecule or the vector, more particularly heterologously expressing the nucleic acid sequence or with recombinant protein application. A non-limiting example of a host cell is a HEK cell.
SEQ ID No. 7 (cDNA of LRR domain from LRRC37B)
ATCTTCACCACCTTAAATTTCCAAGGAAACTATATTTCATACCTTGATGGAAATGTATGGAAAGCATACAGTTGGA CCGAGAAACTAATTCTCAGTGAAAATTATTTGACTGAATTACCTAAGGATTCATTTGAAGGCCTGCTATACCTCCA GTATTTAGATTTATCCTGCAATAAAATACGATATATTGAAAGACAAACATTTGAATCACTACCATTTTTGCAGTAT ATAAATCTGGGCTGCAATTTAATTACAAAACTGAGCCTTGGAACATTTCAGGCCTGGCACGGAATGCAGTTTTTA CACAACTTAATTCTCAATCGCAATCCTCTGACTACTGTCGAAGATCCATATCTCTTTGAACTGCCGGCATTAAAATA TCTAGACATGGGAACAACACACATCACACTTACAACACTTAAGAACATTCTC
SEQ ID No. 8 (amino acid sequence of LRR domain from LRRC37B)
IFTTLNFQGNYISYLDGNVWKAYSWTEKLILSENYLTELPKDSFEGLLYLQYLDLSCNKIRYIERQTFESLPFLQYINLGCNL ITKLSLGTFQAWHGMQFLHNLILNRNPLTTVEDPYLFELPALKYLDMGTTHITLTTLKNIL
In another independent aspect, the present application concerns an isolated LRRC37B protein fragment, preferably an isolated LRRC37B fragment comprising SEQ ID No. 8 or SEQ ID No. 12, a peptidomimetic thereof or a nucleic acid molecule encoding said LRRC37B protein fragment, preferably LRRC37B comprising SEQ ID No. 8 or SEQ ID No. 12 for use as a medicine. Said LRRC37B protein fragment, preferably LRRC37B comprising SEQ ID No. 8 or SEQ ID No. 12 were found to be particularly efficient in modulating the neuronal excitability of human cortical pyramidal neurons, for example to decrease neuron excitability, even more particularly for use to treat seizures, epilepsy, epileptogenesis, migraine, ataxia, autism spectrum disorder, intellectual disability, cardiac arrythmia or pain.
Any of the FGF13A or LRRC37B proteins or protein fragments thereof or homologues thereof as described herein or nucleic acid molecules encoding them or vectors comprising the nucleic acid molecules are provided for use as a medicine, more particularly for use to modulate neuron excitability, even more particularly for use to decrease neuron excitability, even more particularly for use to treat seizures, epilepsy or epileptogenesis.
Also any of the peptidomimetics or fusion proteins or pharmaceutical compositions disclosed herein is provided for use as a medicine, more particularly for use to modulate neuron excitability, even more particularly for use to decrease neuron excitability, even more particularly for use to treat seizures, epilepsy or epileptogenesis.
Drug administration across the blood-brain barrier
The blood-brain barrier (BBB) is a protective layer of tightly joined cells that lines the blood vessels of the brain which prevents entry of harmful substances (e.g. toxins, infectious agents) and restricts entry of (non-lipid) soluble molecules that are not recognized by specific transport carriers into the brain. This poses a challenge in the delivery of drugs, such as the FGF13A derived peptides or peptidomimetics or the LRRC37B derived peptides or peptidomimetics as described herein, to the central nervous system or brain in that drugs transported by the blood not necessarily will pass the blood-brain barrier. Several options are nowadays available for delivery of drugs across the BBB (Peschillo et al. 2016, J Neurointervent Surg 8:1078-1082; Miller & O'Callaghan 2017, Metabolism 69:S3-S7; Drapeau & Fortin 2015, Current Cancer Drug Targets 15:752-768).
Drugs can be directly injected into the brain (invasive strategy) or can be directed into the brain after BBB disruption with a pharmacological agent (pharmacologic strategy). Invasive means of BBB disruption are associated with the risk of hemorrhage, infection or damage to diseased and normal brain tissue from the needle or catheter. Direct drug deposition may be improved by the technique of convection- enhanced delivery. Longer term delivery of a therapeutic protein can be achieved by implantation of genetically modified stem cells, by recombinant viral vectors, by means of osmotic pumps, or by means of incorporating the therapeutic drug in a polymer (slow release; can be implanted locally).
Pharmacologic BBB disruption has the drawback of being non-selective and can be associated with unwanted effects on blood pressure and the body's fluid balance. This is circumvented by targeted or selective administration of the pharmacologic BBB disrupting agent. As an example, intra-arterial cerebral infusion of an antibody (bevacizumab) in a brain tumor was demonstrated after osmotic disruption of the BBB with mannitol (Boockvar et al. 2011, J Neurosurg 114:624-632); other agents capable of disrupting the BBB pharmacologically include bradykinin and leukotriene C4 (e.g. via intracarotid infusion; Nakano et al. 1996, Cancer Res 56:4027-4031).
BBB transcytosis and efflux inhibition are other strategies to increase brain uptake of drugs supplied via the blood. Using transferrin or transferrin-receptor antibodies as carrier of a drug is one example of exploiting a natural BBB transcytosis process (Friden et al. 1996, J Pharmacol Exp Ther 278:1491-1498). Exploiting BBB transcytosis for drug delivery is also known as the molecular Trojan horse strategy. Another mechanism underlying BBB, efflux pumps or ATP-binding cassette (ABC) transporters (such as breast cancer resistance protein (BCRP/ABCG2) and P-glycoprotein (Pgp/MDRl/ABCBl)), can be blocked in order to increase uptake of compounds (e.g. Carcaboso et al. 2010, Cancer Res 70:4499-4508). Therapeutic drugs can alternatively be loaded in liposomes to enhance their crossing of the BBB, an approach also known as liposomal Trojan horse strategy.
A more recent and promising avenue for delivering therapeutic drugs to the brain consists of (transient) BBB disruption by means of ultrasound, more particularly focused ultrasound (FUS; Miller et al. 2017, Metabolism 69:S3-S7). Besides being non-invasive, this technique has, often in combination with realtime imaging, the advantage of precise targeting to a diseased area of the brain. Therapeutic drugs can be delivered in e.g. microbubbles e.g. stabilized by an albumin or other protein, a lipid, or a polymer. Therapeutic drugs can alternatively, or in conjunction with microbubbles, be delivered by any other method, and subsequently FUS can enhance local uptake of any compound present in the blood (e.g. Nance et al. 2014, J Control Release 189:123-132). Just one example is that of FUS-assisted delivery of antibodies directed against toxic amyloid-beta peptide with demonstration of reduced pathology in mice (Jordao et al. 2010, PloS One 5:el0549). Microbubbles with a therapeutic drug load can also be induced to burst (hyperthermic effect) in the vicinity of the target cells by means of FUS, and when driven by e.g. a heat shock protein gene promoter, localized temporary expression of a therapeutic protein can be induced by ultrasound hyperthermia (e.g. Lee Titsworth et al. 2014, Anticancer Res 34:565-574). Alternatives for ultrasound to induce the hyperthermia effect are microwaves, laser-induced interstitial thermotherapy, and magnetic nanoparticles (e.g. Lee Titsworth et al. 2014, Anticancer Res 34:565-574).
Screening methods to identify modulators of the LRRC37B-FGF13A-NAV1.6 interaction
In a third aspect, screening methods are provided to identify modulators of the LRRC37B-FGF13A- NAV1.6 complex or alternatively phrased to identify modulators of neuronal excitability.
In a first embodiment, the modulator increases or reduces the LRRC37B-FGF13A interaction compared to a situation in the absence of the modulator. The application thus provides a method, more particularly an in vitro method, of identifying a modulator of neuronal excitability or a modulator of the LRRC37B- FGF13A interaction, said method comprises the steps of: i) providing the LRRC37B protein, or a fragment thereof comprising or consisting of the LRR domain as depicted in SEQ ID No. 8; ii) providing the FGF13A protein or a fragment thereof comprising or consisting of the fragment as depicted in SEQ ID No. 15 or SEQ ID No. 4; iii) providing a test compound that is candidate for being a modulator of neuronal excitability or of the LRRC37B-FGF13A interaction; iv) contacting LRRC37B or the fragment thereof provided in i) with the FGF13A protein or the fragment thereof provided in ii) in the presence or absence of the test compound provided in iii); v) identifying as modulator of neuronal excitability or of the LRRC37B-FGF13A interaction, from iv) the test compound that is, compared to identical conditions but in absence of said test compound, statistically significantly reducing or increasing the binding of the FGF13A protein or the fragment thereof to LRRC37B or the fragment thereof.
In other words, said test compound can be considered as modulator of the LRRC37B-FGF13A interaction if said test compound can statistically significantly reduce or increase the binding of the FGF13A protein or the fragment thereof to LRRC37B or the fragment thereof, compared to the identical conditions in absence of said test compound.
In one embodiment, the test compound that reduces the binding between FGF13A and LRRC37B protein or fragments thereof shows a higher affinity to LRRC37B than FGF13A and therefore competes with FGF13A for binding to LRRC37B. In a particular embodiment, the test compound is a peptide consisting of 40, 30, 20, 15 or 10 contiguous amino acids or less, wherein the peptide is a fragment of FGF13A-S fragment as depicted SEQ ID No. 4, preferably the fragment as depicted in SEQ ID No. 15 or the test compound is a peptidomimetic of said peptide.
In another embodiment, the binding of LRRC37B or the fragment thereof to the FGF13A protein or the fragment thereof is determined by immunologic, fluorescent or radiologic detection, co-sedimentation, co-immunoprecipitation, or electron microscopy. Assays for studying receptor binding are well-known by the person skilled in the art. A non-limiting example is the fluorescence polarization (FP) assay. The assay is based on the rotational movement of fluorescently labelled molecules in solution. Unbound molecules rotate rapidly, are therefore randomly orientated prior to light emission and hence show a low polarization value. However, if the rotation of a fluorescently labelled molecule is slowed down because it binds to a large complex, it shows a high polarization value. In another particular embodiment, the LRRC37B or the fragment thereof in step i) is provided by one or more cells expressing LRRC37B or the fragment thereof. In another particular embodiment, the FGF13A or the fragment thereof in step ii) is administered extracellularly to the one or more cells expressing LRRC37B or the fragment thereof.
In a preferred embodiment, the present invention relates to an in vitro method of identifying an LRRC37B binding peptide, said method comprises the steps of: i) Providing a LRRC37B protein, or a fragment thereof comprising or consisting of a LRR domain comprising or consisting of SEQ ID No. 8; ii) providing a FGF13A protein fragment derived from a FGF13A S-fragment, preferably the FGF13A fragment comprising or consisting of SEQ ID No. 15 or SEQ ID No. 4 or generating a peptidomimetic derived from a FGF13A S-fragment, preferably the FGF13A fragment comprising or consisting of SEQ ID No. 15 or SEQ ID No. 4; iii) contacting the LRRC37B protein or the fragment thereof provided in i) with the FGF13A protein fragment or peptidomimetic provided in ii); identifying as the LRRC37B binding peptide from iii) a FGF13A protein fragment or peptidomimetic thereof that shows a statistically significantly increased binding to the LRRC37B protein or the fragment thereof provided in step i) compared to the binding between the FGF13A fragment comprising or consisting of SEQ ID No. 15 or SEQ ID No. 4 and the LRRC37B protein or the fragment thereof.
In a particular embodiment, a method is provided of identifying a modulator of neuronal excitability or a LRRC37B binding peptide, said method comprises the steps of: i) providing the LRRC37B protein, or a fragment thereof comprising or consisting of the LRR domain as depicted in SEQ ID No. 8; ii) providing a plurality of FGF13A protein fragments derived from the FGF13A S-fragment as depicted in SEQ ID No. 4 or SEQ ID No. 15 or generating a plurality of peptidomimetics derived from the FGF13A S-fragment as depicted in SEQ ID No. 4 or SEQ ID No. 15; iii) contacting LRRC37B or the fragment thereof provided in i) with the FGF13A protein fragment or peptidomimetics provided in ii); iv) identifying as modulator of neuronal excitability or as LRRC37B binding peptide from iii) a FGF13A protein fragment or peptidomimetic that shows a statistically significantly increased binding to LRRC37B or the fragment thereof compared to the binding between the fragment as depicted in SEQ ID No. 4 or SEQ ID No. 15 and LRRC37B or the fragment thereof.
Preferably, in said methods according to the present invention, in particular the methods according to the third aspect, the binding of the LRRC37B protein or the fragment thereof to the FGF13A protein or the fragment thereof is determined by immunologic or radiologic detection, co-sedimentation, coimmunoprecipitation or electron microscopy. In another embodiment, said binding may be determined by any suitable method known in the prior art without departing from the scope of the present invention. Preferably, the LRRC37B protein or the fragment thereof can be provided by cells expressing the LRRC37B protein or the fragment thereof, preferably LRRC37B protein or the fragment thereof is provided by cells expressing the LRRC37B protein or the fragment thereof.
Preferably, the FGF13A protein or the fragment or peptidomimetic thereof can be administered extracellularly to the cells expressing the LRRC37B protein or the fragment thereof, preferably the FGF13A protein or the fragment or peptidomimetic thereof is administered extracellularly to the cells expressing the LRRC37B protein or the fragment thereof.
In the Example section it is demonstrated that FGF13A binds NAVI.6 and inhibits the latter's activity. NAVI.6 (UniProt ID: Q9UQD0; also termed SCN8A or PN4) is a voltage-gated sodium channel a subunit that is the most abundantly expressed isoform in the CNS during adulthood and is enriched at the axon initial segment and at the nodes of Ranvier. The channels are highly concentrated in sensory and motor axons in the peripheral nervous system. NAVI.6 facilitates action potential propagation when the membrane potential is depolarized by an influx of Na+ ions. However, NAVI.6 is able to sustain repetitive excitation and firing. The high frequency firing characteristic of NAVI.6 is caused by a persistent and resurgent sodium current. This characteristic is caused by slow activation of the sodium channel following repolarization, which allows a steady-state sodium current after the initial action potential propagation. NAVI.6 is tetradotoxin (TTX)-sensitive. The cDNA sequence of NAVI.6 is depicted in SEQ ID No. 13 and its amino acid sequence in SEQ ID No. 14.
SEQ ID No. 13 (cDNA sequence of NAV1.6)
ATGGCAGCGCGGCTGCTTGCACCACCAGGCCCTGATAGTTTCAAGCCTTTCACCCCTGAGTCACTGGCAAACATT GAGAGGCGCATTGCTGAGAGCAAGCTCAAGAAACCACCAAAGGCCGATGGCAGTCATCGGGAGGACGATGAG GACAGCAAGCCCAAGCCAAACAGCGACCTGGAAGCAGGGAAGAGTTTGCCTTTCATCTACGGGGACATCCCCCA AGGCCTGGTTGCAGTTCCCCTGGAGGACTTTGACCCATACTATTTGACGCAGAAAACCTTTGTAGTATTAAACAG AGGGAAAACTCTCTTCAGATTTAGTGCCACGCCTGCCTTGTACATTTTAAGTCCTTTTAACCTGATAAGAAGAATA GCTATTAAAATTTTGATACATTCAGTATTTAGCATGATCATTATGTGCACTATTTTGACCAACTGTGTATTCATGAC TTTTAGTAACCCTCCTGACTGGTCGAAGAATGTGGAGTACACGTTCACAGGGATTTATACATTTGAATCACTAGT GAAAATCATTGCAAGAGGTTTCTGCATAGATGGCTTTACCTTTTTACGGGACCCATGGAACTGGTTAGATTTCAG TGTCATCATGATGGCGTATATAACAGAGTTTGTAAACCTAGGCAATGTTTCAGCTCTACGCACTTTCAGGGTACTG AGGGCTTTGAAAACTATTTCGGTAATCCCAGGCCTGAAGACAATTGTGGGTGCCCTGATTCAGTCTGTGAAGAAA CTGTCAGATGTGATGATCCTGACAGTGTTCTGCCTGAGTGTTTTTGCCTTGATCGGACTGCAGCTGTTCATGGGG
AACCTTCGAAACAAGTGTGTTGTGTGGCCCATAAACTTCAACGAGAGCTATCTTGAAAATGGCACCAAAGGCTTT
GATTGGGAAGAGTATATCAACAATAAAACAAATTTCTACACAGTTCCTGGCATGCTGGAACCTTTACTCTGTGGG
AACAGTTCTGATGCTGGGCAATGCCCAGAGGGATACCAGTGTATGAAAGCAGGAAGGAACCCCAACTATGGTTA
CACAAGTTTTGACACTTTTAGCTGGGCCTTCTTGGCATTATTTCGCCTTATGACCCAGGACTATTGGGAAAACTTG
TATCAATTGACTTTACGAGCAGCCGGGAAAACATACATGATCTTCTTCGTCTTGGTCATCTTTGTGGGTTCTTTCTA
TCTGGTGAACTTGATCTTGGCTGTGGTGGCCATGGCTTATGAAGAACAGAATCAGGCAACACTGGAGGAGGCAG
AACAAAAAGAGGCTGAATTTAAAGCAATGTTGGAGCAACTTAAGAAGCAACAGGAAGAGGCACAGGCTGCTGC
GATGGCCACTTCAGCAGGAACTGTCTCAGAAGATGCCATAGAGGAAGAAGGTGAAGAAGGAGGGGGCTCCCCT
CGGAGCTCTTCTGAAATCTCTAAACTCAGCTCAAAGAGTGCAAAGGAAAGACGTAACAGGAGAAAGAAGAGGA
AGCAAAAGGAACTCTCTGAAGGAGAGGAGAAAGGGGATCCCGAGAAGGTGTTTAAGTCAGAGTCAGAAGATG
GCATGAGAAGGAAGGCCTTTCGGCTGCCAGACAACAGAATAGGGAGGAAATTTTCCATCATGAATCAGTCACTG
CTCAGCATCCCAGGCTCGCCCTTCCTCTCCCGCCACAACAGCAAGAGCAGCATCTTCAGTTTCAGGGGACCTGGG
CGGTTCCGAGACCCGGGCTCCGAGAATGAGTTCGCGGATGACGAGCACAGCACGGTGGAGGAGAGCGAGGGC
CGCCGGGACTCCCTCTTCATCCCCATCCGGGCCCGCGAGCGCCGGAGCAGCTACAGCGGCTACAGCGGCTACAG
CCAGGGCAGCCGCTCCTCGCGCATCTTCCCCAGCCTGCGGCGCAGCGTGAAGCGCAACAGCACGGTGGACTGCA
ACGGCGTGGTGTCCCTCATCGGCGGCCCCGGCTCCCACATCGGCGGGCGTCTCCTGCCAGAGGCTACAACTGAG
GTGGAAATTAAGAAGAAAGGCCCTGGATCTCTTTTAGTTTCCATGGACCAATTAGCCTCCTACGGGCGGAAGGA
CAGAATCAACAGTATAATGAGTGTTGTTACAAATACACTAGTAGAAGAACTGGAAGAGTCTCAGAGAAAGTGCC
CGCCATGCTGGTATAAATTTGCCAACACTTTCCTCATCTGGGAGTGCCACCCCTACTGGATAAAACTGAAAGAGA
TTGTGAACTTGATAGTTATGGACCCTTTTGTGGATTTAGCCATCACCATCTGCATCGTCCTGAATACACTGTTTATG
GCAATGGAGCACCATCCTATGACACCACAATTTGAACATGTCTTGGCTGTAGGAAATCTGGTAAGTATGAAATTC
AGGGATACGGCATATTTGCCAAATAGTGGAAATGTGAAGTACTGACAAAACTTTTCCCTTTTTCCCTCTCATAGGT
TTTCACTGGAATTTTCACAGCGGAAATGTTCCTGAAGCTCATAGCCATGGATCCCTACTATTATTTCCAAGAAGGT
TGGAACATTTTTGACGGATTTATTGTCTCCCTCAGTTTAATGGAACTGAGTCTAGCAGACGTGGAGGGGCTTTCA
GTGCTGCGATCTTTCCGATTGCTCCGAGTCTTCAAATTGGCCAAATCCTGGCCCACCCTGAACATGCTAATCAAGA
TTATTGGAAATTCAGTGGGTGCCCTGGGCAACCTGACACTGGTGCTGGCCATTATTGTCTTCATCTTTGCCGTGGT
GGGGATGCAACTCTTTGGAAAAAGCTACAAAGAGTGTGTCTGCAAGATCAACCAGGACTGTGAACTCCCTCGCT
GGCATATGCATGACTTTTTCCATTCCTTCCTCATTGTCTTTCGAGTGTTGTGCGGGGAGTGGATTGAGACCATGTG
GGACTGCATGGAAGTGGCAGGCCAGGCCATGTGCCTCATTGTCTTTATGATGGTCATGGTGATTGGCAACTTGG
TGGTGCTGAACCTGTTTCTGGCCTTGCTCCTGAGCTCCTTCAGTGCAGACAACCTGGCTGCCACAGATGACGATG
GGGAAATGAACAACCTCCAGATCTCAGTGATCCGTATCAAGAAGGGTGTGGCCTGGACCAAACTAAAGGTGCAC
GCCTTCATGCAGGCCCACTTTAAGCAGCGTGAGGCTGATGAGGTGAAGCCTCTGGATGAGTTGTATGAAAAGAA
GGCCAACTGTATCGCCAATCACACCGGTGCAGACATCCACCGGAATGGTGACTTCCAGAAGAATGGCAATGGCA CAACCAGCGGCATTGGCAGCAGCGTGGAGAAGTACATCATTGATGAGGACCACATGTCCTTCATCAACAACCCC
AACTTGACTGTACGGGTACCCATTGCTGTGGGCGAGTCTGACTTTGAGAACCTCAACACAGAGGATGTTAGCAG
CGAGTCGGATCCTGAAGGCAGCAAAGATAAACTAGATGACACCAGCTCCTCTGAAGGAAGCACCATTGATATCA
AACCAGAAGTAGAAGAGGTCCCTGTGGAACAGCCTGAGGAATACTTGGATCCAGATGCCTGCTTCACAGAAGGT
TGTGTCCAGCGGTTCAAGTGCTGCCAGGTCAACATCGAGGAAGGGCTAGGCAAGTCTTGGTGGATCCTGCGGAA
AACCTGCTTCCTCATCGTGGAGCACAACTGGTTTGAGACCTTCATCATCTTCATGATTCTGCTGAGCAGTGGCGCC
CTGGCCTTCGAGGACATCTACATTGAGCAGAGAAAGACCATCCGCACCATCCTGGAATATGCTGACAAAGTCTTC
ACCTATATCTTCATCCTGGAGATGTTGCTCAAGTGGACAGCCTATGGCTTCGTCAAGTTCTTCACCAATGCCTGGT
GTTGGCTGGACTTCCTCATTGTGGCTGTCTCTTTAGTCAGCCTTATAGCTAATGCCCTGGGCTACTCGGAACTAGG
TGCCATAAAGTCCCTTAGGACCCTAAGAGCTTTGAGACCCTTAAGAGCCTTATCACGATTTGAAGGGATGAGGGT
GGTGGTGAATGCCTTGGTGGGCGCCATCCCCTCCATCATGAATGTGCTGCTGGTGTGTCTCATCTTCTGGCTGAT
TTTCAGCATCATGGGAGTTAACTTGTTTGCGGGAAAGTACCACTACTGCTTTAATGAGACTTCTGAAATCCGATTT
GAAATTGAAGATGTCAACAATAAGACCGAATGTGAAAAGCTTATGGAGGGGAACAATACAGAGATCAGATGGA
AGAACGTGAAGATCAACTTTGACAATGTTGGGGCAGGATACCTGGCCCTTCTTCAAGTAGCAACCTTCAAAGGCT
GGATGGACATCATGTATGCAGCTGTAGATTCCCGGAAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGAC
CAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATC
CACTTTGCCTTTCTCTCCACAGCCTGATGAGCAGCCTAAGTATGAGGACAATATCTACATGTACATCTATTTTGTCA
TCTTCATCATCTTCGGCTCCTTCTTCACCCTGAACCTGTTCATTGGTGTCATCATTGATAACTTCAATCAACAAAAG
AAAAAGTTCGGAGGTCAGGACATCTTCATGACCGAAGAACAGAAGAAGTACTACAATGCCATGAAAAAGCTGG
GCTCAAAGAAGCCACAGAAACCTATTCCCCGCCCCTTGAACAAAATCCAAGGAATCGTCTTTGATTTTGTCACTCA
GCAAGCCTTTGACATTGTTATCATGATGCTCATCTGCCTTAACATGGTGACAATGATGGTGGAGACAGACACTCA
AAGCAAGCAGATGGAGAACATCCTCTACTGGATTAACCTGGTGTTTGTTATCTTCTTCACCTGTGAGTGTGTGCTC
AAAATGTTTGCGTTGAGGCACTACTACTTCACCATTGGCTGGAACATCTTCGACTTCGTGGTAGTCATCCTCTCCA
TTGTGGGAATGTTCCTGGCAGATATAATTGAGAAATACTTTGTTTCCCCAACCCTATTCCGAGTCATCCGATTGGC
CCGTATTGGGCGCATCTTGCGTCTGATCAAAGGCGCCAAAGGGATTCGTACCCTGCTCTTTGCCTTAATGATGTC
CTTGCCTGCCCTGTTCAACATCGGCCTTCTGCTCTTCCTGGTCATGTTCATCTTCTCCATTTTTGGGATGTCCAATTT
TGCATATGTGAAGCACGAGGCTGGTATCGATGACATGTTCAACTTTGAGACATTTGGCAACAGCATGATCTGCCT
GTTTCAAATCACAACCTCAGCTGGTTGGGATGGCCTGCTGCTGCCCATCCTAAACCGCCCCCCTGACTGCAGCCTA
GATAAGGAACACCCAGGGAGTGGCTTTAAGGGAGATTGTGGGAACCCCTCAGTGGGCATCTTCTTCTTTGTAAG
CTACATCATCATCTCTTTCCTAATTGTCGTGAACATGTACATTGCCATCATCCTGGAGAACTTCAGTGTAGCCACA
GAGGAAAGTGCAGACCCTCTGAGTGAGGATGACTTTGAGACCTTCTATGAGATCTGGGAGAAGTTCGACCCCGA
TGCCACCCAGTTCATTGAGTACTGTAAGCTGGCAGACTTTGCAGATGCCTTGGAGCATCCTCTCCGAGTGCCCAA
GCCCAATACCATTGAGCTCATCGCTATGGATCTGCCAATGGTGAGCGGGGATCGCATCCACTGCTTGGACATCCT
TTTTGCCTTCACCAAGCGGGTCCTGGGAGATAGCGGGGAGTTGGACATCCTGCGGCAGCAGATGGAAGAGCGG TTCGTGGCATCCAATCCTTCCAAAGTGTCTTACGAGCCAATCACAACCACACTGCGTCGCAAGCAGGAGGAGGTA
TCTGCAGTGGTCCTGCAGCGTGCCTACCGGGGACATTTGGCAAGGCGGGGCTTCATCTGCAAAAAGACAACTTC
TAATAAGCTGGAGAATGGAGGCACACACCGGGAGAAAAAAGAGAGCACCCCATCTACAGCCTCCCTCCCGTCCT
ATGACAGTGTAACTAAACCTGAAAAGGAGAAACAGCAGCGGGCAGAGGAAGGAAGAAGGGAAAGAGCCAAAA
GACAAAAAGAGGTCAGAGAATCCAAGTGTTGA
SEQ ID No. 14 (amino acid sequence of NAVI.6)
MAARLLAPPGPDSFKPFTPESLANIERRIAESKLKKPPKADGSHREDDEDSKPKPNSDLEAGKSLPFIYGDIPQGLVAVP
LEDFDPYYLTQKTFVVLNRGKTLFRFSATPALYILSPFNLIRRIAIKILIHSVFSM IIMCTILTNCVFMTFSNPPDWSKNVEY
TFTGIYTFESLVKIIARGFCIDGFTFLRDPWNWLDFSVIMMAYITEFVNLGNVSALRTFRVLRALKTISVIPGLKTIVGALI
QSVKKLSDVMILTVFCLSVFALIGLQLFMGNLRNKCVVWPINFNESYLENGTKGFDWEEYINNKTNFYTVPGMLEPLL
CGNSSDAGQCPEGYQCM KAGRNPNYGYTSFDTFSWAFLALFRLMTQDYWENLYQLTLRAAGKTYM IFFVLVIFVGS
FYLVNLILAVVAMAYEEQNQATLEEAEQKEAEFKAMLEQLKKQQEEAQAAAMATSAGTVSEDAIEEEGEEGGGSPRS
SSEISKLSSKSAKERRNRRKKRKQKELSEGEEKGDPEKVFKSESEDGMRRKAFRLPDNRIGRKFSIMNQSLLSIPGSPFLS
RHNSKSSIFSFRGPGRFRDPGSENEFADDEHSTVEESEGRRDSLFIPIRARERRSSYSGYSGYSQGSRSSRIFPSLRRSVKR
NSTVDCNGVVSLIGGPGSHIGGRLLPEATTEVEIKKKGPGSLLVSMDQLASYGRKDRINSIMSVVTNTLVEELEESQRKC
PPCWYKFANTFLIWECHPYWIKLKEIVNLIVMDPFVDLAITICIVLNTLFMAMEHHPMTPQFEHVLAVGNLVFTGIFTA
EM FLKLIAMDPYYYFQEGWNIFDGFIVSLSLMELSLADVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNL
TLVLAIIVFIFAVVGMQLFGKSYKECVCKINQDCELPRWHMHDFFHSFLIVFRVLCGEWIETMWDCMEVAGQAMCLI
VFMMVMVIGNLVVLNLFLALLLSSFSADNLAATDDDGEM NNLQISVIRIKKGVAWTKLKVHAFMQAHFKQREADEV
KPLDELYEKKANCIANHTGADIHRNGDFQKNGNGTTSGIGSSVEKYIIDEDHMSFINNPNLTVRVPIAVGESDFENLNT
EDVSSESDPEGSKDKLDDTSSSEGSTIDIKPEVEEVPVEQPEEYLDPDACFTEGCVQRFKCCQ.VNIEEGLGKSWWILRKT
CFLIVEHNWFETFIIFM ILLSSGALAFEDIYIEQRKTIRTILEYADKVFTYIFILEMLLKWTAYGFVKFFTNAWCWLDFLIVA
VSLVSLIANALGYSELGAIKSLRTLRALRPLRALSRFEGMRVVVNALVGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKYH
YCFNETSEIRFEIEDVNNKTECEKLMEGNNTEIRWKNVKINFDNVGAGYLALLQVATFKGWM DIMYAAVDSRKPDE
QPKYEDNIYMYIYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKFGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPLNKI
QGIVFDFVTQQAFDIVIMM LICLNMVTM MVETDTQSKQM ENILYWINLVFVIFFTCECVLKMFALRHYYFTIGWNIF
DFVVVILSIVGMFLADIIEKYFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNIGLLLFLVM FIFSIFGMSNF
AYVKHEAGIDDMFNFETFGNSM ICLFQITTSAGWDGLLLPILNRPPDCSLDKEHPGSGFKGDCGNPSVGIFFFVSYIIISF
LIVVNMYIAIILENFSVATEESADPLSEDDFETFYEIWEKFDPDATQFIEYCKLADFADALEHPLRVPKPNTIELIAMDLP
MVSGDRIHCLDILFAFTKRVLGDSGELDILRQQMEERFVASNPSKVSYEPITTTLRRKQEEVSAVVLQRAYRGHLARRG
FICKKTTSNKLENGGTHREKKESTPSTASLPSYDSVTKPEKEKQQRAEEGRRERAKRQKEVRESKC
In a preferred embodiment of the third aspect, the modulator of neuronal excitability as provided herein increases or reduces the FGF13A-NAV1.6 interaction. The application thus provides a method, more particularly an in vitro method, of identifying a modulator of neuronal excitability or a modulator of the
FGF13A-NAV1.6 interaction, said method comprises the steps of: i) providing the FGF13A protein, or a fragment thereof comprising or consisting of the S protein fragment as depicted in SEQ ID No. 4, preferably as depicted in SEQ ID No. 15, or a peptidomimetic thereof; ii) providing the NAVI.6 protein as depicted in SEQ ID No. 14 or a fragment thereof interacting with the FGF13A protein or fragment thereof; iii) providing a test compound that is candidate for being a modulator of neuronal excitability or of the FGF13A-NAV1.6 interaction; iv) contacting FGF13A or the fragment thereof provided in i) with the NAVI.6 protein or fragment thereof provided in ii) in the presence or absence of the compound provided in iii); v) identifying as modulator of neuronal excitability or of the FGF13A-NAV1.6 interaction, from iv) the test compound that is, compared to identical conditions but in absence of the test compound, statistically significantly reducing or increasing the binding of the FGF13A protein or fragment thereof to NAVI.6 or the fragment thereof.
In specific embodiments, immune-based assays are comprised in the claimed method, more specifically "immune-based assays" or "immune-based detection" for monitoring the binding between protein fragments or between compounds and one or more proteins. In said embodiments, "immune-based assays" comprise the most broadly used bio-detection technologies that are based on the use of antibodies, and are well known in the art. Antibodies are highly suited for detecting small quantities of target proteins in the presence of complex mixtures of proteins. As used herein, an "immune-based assay", "immunoassay" or "immune-based detection" (each of these terms can be used interchangeably) refers to a biochemical binding assay involving binding between antibodies and antigen, which measures the presence or concentration of a substance in a sample, such as a biological sample, using the reaction of an antibody to its cognate antigen, for example the specific binding of an antibody to a protein. Both the presence of the antigen or the amount of the antigen present can be measured.
Examples of immunoassays are enzyme linked immunosorbent assays (ELISAs), enzyme linked immunospot assay (ELISPOT), immunobead capture assays, Western blotting, gel-shift assays, protein arrays, multiplexed bead arrays, magnetic capture, fluorescence resonance energy transfer (FRET), a sandwich assay, a competitive assay, an immunoassay using a biosensor, an immunoprecipitation assay etc. Examples of assays which can require these detection methods for producing compounds in the context of the present invention are described in the Example section, without the purpose of being limitative. It should be clear to the skilled artisan that the present screening methods might be based on a combination or a series of measurements, particularly when establishing the link between the impairment of the activity of the NAVI.6 channel by specific test compounds. Also, it should be clear that there is no specific order in performing these measurements while practicing the present invention.
In general, immune-based assays involve contacting a sample suspected of containing a molecule of interest (such as the test compound) with an antibody to the molecule of interest or contacting an antibody to a molecule of interest with a molecule that can be bound by the antibody, as the case may be, under conditions effective to allow the formation of immune complexes. Contacting a sample with the antibody to the molecule of interest or with the molecule that can be bound by an antibody to the molecule of interest under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply bringing into contact the molecule or antibody and the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any molecules (e.g., antigens) present to which the antibodies can bind. In many forms of immunoassay, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, can then be washed to remove any non- specif ically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
Immunoassays can include methods for detecting or quantifying the amount of a molecule of interest in a sample, which methods generally involve the detection or quantitation of any immune complexes formed during the binding process. In general, the immune-based detection is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or any other known label. See, for example, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each of which is incorporated herein by reference in its entirety and specifically for teachings regarding immune-based detection methods and labels. As used herein, a label can include a fluorescent dye, a member of a binding pair, such as biotin/streptavidin, a metal (e.g., gold), or an epitope tag that can specifically interact with a molecule that can be detected, such as by producing a coloured substrate or fluorescence. Substances suitable for detectably labelling proteins include fluorescent dyes (also known herein as fluorochromes and fluorophores) and enzymes that react with colorimetric substrates (e.g., horseradish peroxidase). The use of fluorescent dyes is generally preferred in the practice of the invention as they can be detected at very low amounts. Furthermore, in the case where multiple antigens are reacted with a single array, each antigen can be labelled with a distinct fluorescent compound for simultaneous detection. Labelled spots on the array are detected using a fluorimeter, the presence of a signal indicating an antigen bound to a specific antibody. Fluorophores are compounds or molecules that luminesce. Typically, fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength.
A variety of immunoassays can be used to detect one or more of the proteins disclosed or incorporated by reference herein. ELISA is a heterogeneous immunoassay, which can be used in the methods disclosed herein. The assay can be used to detect protein antigens in various formats. In the "sandwich" format the antigen being assayed is held between two different antibodies. In this method, a solid surface is first coated with a solid phase antibody. The in vitro system composition comprises the antigen, to which the test compound is added, allows binding of the test compound, and therefore reduces the detection of the antigen via reaction with the bound antibody. Any unbound antigen is washed away. A known amount of enzyme-labelled antibody is then allowed to react with the bound antigen. Any excess unbound enzyme-linked antibody is washed away after the reaction. The substrate for the enzyme used in the assay is then added and the reaction between the substrate and the enzyme produces a colour change. The amount of visual colour change is a direct measurement of specific enzyme-conjugated bound antibody, and consequently the antigen present in the sample tested. ELISA can also be used as a competitive assay. In the competitive assay format, the test specimen containing the antigen to be determined is mixed with a precise amount of enzyme-labelled antigen and both compete for binding to an anti-antigen antibody attached to a solid surface. Excess free enzyme-labelled antigen is washed off before the substrate for the enzyme is added. The amount of colour intensity resulting from the enzymesubstrate interaction is a measure of the amount of antigen in the sample tested. A heterogeneous immunoassay, such as an ELISA, can be used to detect any of the proteins disclosed or incorporated by reference herein. In many immunoassays, as described elsewhere herein, detection of antigen is made with the use of antigens specific antibodies as detector molecules. However, immunoassays and the systems and methods of the present invention are not limited to the use of antibodies as detector molecules. Any substance that can bind or capture the antigen within a given sample may be used. Aside from antibodies, suitable substances that can also be used as detector molecules include but are not limited to enzymes, peptides, proteins, and nucleic acids. Further, there are many detection methods known in the art in which the captured antigen may be detected. In some assays, enzyme-linked antibodies produce a colour change. In other assays, detection of the captured antigen is made through detecting fluorescent, luminescent, chemiluminescent, or radioactive signals. The system and methods of the current invention is not limited to the particular types of detectable signals produced in an immunoassay.
In a preferred embodiment, a method of identifying a modulator of neuron excitability comprising the steps of: generating a peptide consisting of less than 30 contiguous amino acids wherein the peptide is a fragment of FGF13A S-fragment as depicted in SEQ ID No. 4, preferably said peptide comprising SEQ ID No. 15, or generating a peptidomimetic of said peptide; providing a NAVI.6 protein as depicted in SEQ ID No. 14 or a functional fragment thereof; contacting a NAVI.6 protein or the fragment thereof with said peptide or peptidomimetic; identifying as a modulator of neuron excitability a peptide or peptidomimetic that, compared to identical conditions but in absence of the peptide or peptidomimetic, statistically significantly reduces or increases the activity of the NAVI.6 protein.
Preferably, said NAVI.6 protein or the functional fragment thereof is provided by cells expressing NAVI.6 or the functional fragment thereof. In a preferred embodiment, said cells are electrically excitable cells.
In another embodiment, a method of identifying a modulator of neuron excitability is provided comprising the steps of: i) generating a plurality of FGF13A protein fragments comprising the FGF13A fragment as depicted in SEQ ID No. 15 or SEQ ID No. 4 or derived from the FGF13A fragment as depicted in SEQ ID No. 15 or SEQ ID No. 4 or generating a plurality of peptidomimetics derived from the FGF13A fragment as depicted in SEQ ID No. 15 or SEQ ID No. 4; ii) providing the NAVI.6 protein as depicted in SEQ ID No. 14 or a functional fragment thereof; iii) contacting the NAVI.6 protein provided in ii) with one or more of the FGF13A protein fragment or peptidomimetics provided in step i); iv) identifying as a modulator of neuron excitability from iii) a FGF13A protein fragment or peptidomimetic that, compared to identical conditions but in absence of the FGF13A protein fragment or peptidomimetic, statistically significantly reduces or increases the activity of the NAVI.6.
"Derived from" as used herein means that the FGF13A protein fragment is a part of the FGF13A S- fragment and is thus shorter in length than the FGF13A S-fragment. In some embodiments, said FGF13A protein fragment derived from the FGF13A S-fragment consists of 60, 50, 40, 30, 20, 10 or less contiguous amino acids. In a particular embodiment, the FGF13A protein fragments or peptidomimetics generated in step i) consist of at most 4, 5, 6, 7, 8, 9, 10, 15, 20 or at most 25 amino acids.
Also provided is a method, more particularly an in vitro method, of identifying a modulator of neuronal excitability comprising the steps of: - providing one or more excitable cells expressing the NAVI.6 protein as depicted in SEQ ID No.
14 or a fragment thereof interacting with an FGF13A protein or a fragment thereof comprising or consisting of a fragment as depicted in SEQ ID No. 15 or SEQ ID No. 4;
- contacting the one or more cells with a peptide consisting of less than 60, 50, 40, 30, 25, 20,
15 or 10 contiguous amino acids, wherein the peptide is a fragment of the FGF13A S-fragment as depicted in SEQ ID No. 4 or SEQ ID No. 15 or adding a peptidomimetic of said peptide to the one or more cells;
- identifying the peptide or peptidomimetic as modulator of neuronal excitability if compared to identical conditions but in absence of the peptide or peptidomimetic, the compound statistically significantly reduces or increases the activity of NAVI.6 or the fragment thereof.
In one embodiment, said FGF13A protein fragment, peptide or peptidomimetic shows a higher affinity to NAVI.6 than FGF13A and therefore competes with FGF13A for binding to and thus modulation of NAV1.6.
In another embodiment, the NAVI.6 or the functional fragment thereof (e.g. in step ii)) is provided by cells expressing NAVI.6 or the functional fragment thereof. More particularly, said cells are excitable cells, more particular electrically excitable cells, even more particularly neuron or cardiac cells. In a further embodiment, identifying the modulator of NAVI.6 activity is based on an increased or decreased electrical excitability of the cells.
In principle, assays for determining activity of ion channels are straightforward and there are several fluorescent and non-fluorescent methods available (Birch et al 2004 DDT 9) that are amenable to high- throughput screening in 96-and 384-well plate arrays, as well as higher well densities. Non-fluorescent methods for example directly measure the flux of an ion through the channel of interest and in some cases exploit the non-selective conductance of ions by the channel under investigation. The ion flux can either be measured using radiotracers (e.g. 22Na or [14C]-guanidinium for sodium channels) or atomic absorption spectroscopy (AAS) for the detection of non-radioactive metal ions. Methods have been recently developed that use Li flux for the analysis of sodium channel activity (Birch et al 2004 DDT 9). The advantage of using ion flux measurements is that there is a direct correlation with channel function.
In another embodiment, the above screening method is provided wherein the NAVI.6 voltage-gated sodium channel is provided in a structure that separates a first medium from a second medium, wherein NAVI.6 exhibits independent ion permeation. The amount of the independent ion permeation through the NAVI.6 voltage-gated sodium channel is then measured between the first and second media and compared between presence and absence of a potential modulator of NAVI.6 activity. Hence, a method is provided for identifying a modulator of the NAVI.6 voltage-gated sodium channel or of neuron excitability, the method comprising the following steps: providing the NAVI.6 voltage-gated sodium channel in a structure that separates a first medium from a second medium, wherein NAVI.6 exhibits independent ion permeation; contacting the NAVI.6 voltage-gated sodium channel with a FGF13A derived peptide or peptidomimetic; measuring the amount of the independent ion permeation through the NAVI.6 voltage-gated sodium channel between the first and second media; and comparing the amount of the independent ion permeation measured for the NAVI.6 voltagegated sodium channel contacted with the FGF13A derived peptide or peptidomimetic to the amount of said independent ion permeation measured for the NAVI.6 voltage-gated sodium channel not contacted with the FGF13A derived peptide or peptidomimetic, wherein an increase or decrease in the amount of said independent ion permeation of the NAVI.6 voltage-gated sodium channel contacted with the FGF13A derived peptide or peptidomimetic compared to the voltage-gated sodium channel not contacted with the FGF13A derived peptide or peptidomimetic indicates that the FGF13A derived peptide or peptidomimetic modulates the activity of the voltage-gated sodium channel, more particularly increases or decrease the activity respectively.
In one embodiment, the NAVI.6 activity refers to the ability of NAVI.6 to mediate a sodium current across a membrane. In another embodiment, the FGF13A derived peptide or peptidomimetic is a peptide of at most 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or at most 60 amino acids and consisting of a fragment of the FGF13A S-domain as depicted in SEQ ID No. 4 or a fragment as depicted in SEQ ID No. 15. In another embodiment, the FGF13A derived peptide or peptidomimetic is a peptide consisting of 20, 15 or 10 contiguous amino acids or less, wherein the peptide is a fragment of the FGF13A S-fragment as depicted in SEQ ID No. 15 or SEQ ID No. 4.
In particular embodiments, measuring the amount of independent sodium ion permeation through the voltage-gated sodium channel between the first and second media is by patch-clamp measurement. In other embodiments, measuring the amount of independent sodium ion permeation through the voltagegated sodium channel between the first and second media is by fluorescence measurement. In certain embodiments, measuring the amount of independent sodium ion permeation through the voltage-gated sodium channel between the first and second media is by radiolabeled measurement. In certain embodiments, measuring the amount of independent sodium ion permeation through the voltage-gated sodium channel between the first and second media is by biological assay measurement. Assays can be performed in an in vitro system. Therefore, in one embodiment, said cell expressing a functional NAVI.6 is an in vitro system comprising neuronal cells expressing a functional NAVI.6 channel.
In another embodiment, said cell expressing a functional NAVI.6 channel is selected from a recombinant cell, a neuronal cell or a primary neuron. In a particular embodiment, said cell expressing a functional NAVI.6 channel is a neuron present in an acute brain slice derived from a non-human mammal.
"Permeation" as used herein refers to the rapid and selective transport of (sodium) ions through cell membranes (e.g. through sodium channels) which is essential for initiating action potentials within excitable cells.
An "excitable cell" as used herein refers to a cell or cells with the ability to be electrically excited resulting in the generation of action potentials. Non-limiting examples of excitable cells are neurons, muscle cells (skeletal, cardiac, and smooth), and some endocrine cells (e.g. insulin-releasing pancreatic p cells). Under resting conditions, the membrane potential of an excitable cell, as measured inside relative to outside, is polarized and can, in a non-limiting embodiment, be in the range of about -60mV to about -80mV.
Current application also provides methods for designing, selecting, screening and/or optimizing a chemical entity that binds to and/or modulates the activity of the NAVI.6 voltage-gated sodium channel, said methods comprise the steps of: a. generating a co-crystal comprising the NAVI.6 protein as depicted in SEQ ID No. 14 or fragment thereof and the FGF13A fragment as depicted in SEQ ID No. 15 or SEQ ID No. 4 or generating a co-crystal comprising the LRRC37B fragment as depicted in SEQ ID No. 8 and the FGF13A fragment as depicted in SEQ ID No. 15 or SEQ ID No. 4; b. determining the three-dimensional atomic coordinates of the co-crystal by X-ray diffraction; c. providing the three-dimensional atomic coordinates of the co-crystal on a computer comprising the means for generating three-dimensional structural information from atomic coordinates; and b. designing, selecting, screening and/or optimizing the chemical entity by performing a fitting operation between the chemical entity and the three-dimensional structural information of all or part of said crystal.
The term "fitting operation" refers to an operation that utilizes the atomic coordinates of a chemical entity, binding pocket, molecule or molecular complex, or portion thereof, to associate the chemical entity with the binding pocket, molecule or molecular complex, or portion thereof. This may be achieved by positioning, rotating or translating a chemical entity in the binding pocket to match the shape and electrostatic complementarity of the binding pocket. Covalent interactions, non-covalent interactions such as hydrogen bond, electrostatic, hydrophobic, van der Waals interactions, and non-complementary electrostatic interactions such as repulsive charge-charge, dipole-dipole and charge-dipole interactions may be optimized. Alternatively, one may minimize the deformation energy of binding of the chemical entity to the binding pocket.
Modulating the LRRC37B-SCN1B interaction
In a fourth aspect, the present invention provides a modulator of the interaction between LRRC37B and SCN1B for use as a medicine. Said modulator can be particularly advantageous for use in modulating, i.e. decreasing or increasing the neuronal excitability of human neurons, preferably human cortical pyramidal neurons. In a preferred embodiment, said modulator can be used to decrease neuron excitability, even more particularly for use to treat seizures, epilepsy, epileptogenesis, ataxia, autism spectrum disorder, intellectual disability, cardiac arrythmia or pain.
It is herein demonstrated that LRRC37B binds SCN1B, a NAV p-subunit that modulates VGSCs, more particularly NAVI.6 (Wimmer et al 2010 J Clin Invest 120: 2661-2671). Interestingly, it was found that the LRRC37B competes with NAVI.6 for binding with SCN1B. As such expressing LRRC37B reduces or inhibits the binding between SCN1B and NAVI.6 and therefore decreases neuronal excitability of cortical neurons. Hence, in a fourth aspect the application provides a modulator of neuron excitability or more particularly a modulator of the NAV1.6-SCN1B interaction, wherein the modulator is a LRRC37B protein fragment comprising the B specific domain as depicted in SEQ ID No. 12 or a homologue thereof.
The nucleic acid sequence encoding SCN1B is depicted in SEQ ID No. 9 and the amino acid sequence is depicted in SEQ ID No. 10. SEQ ID No. 11 depicts the nucleic acid sequence encoding the B specific domain of LRRC37B, while SEQ ID No. 12 depicts the amino acid sequence of the B specific domain of LRRC37B.
In a preferred embodiment, an isolated LRRC37B protein fragment comprising or consisting of the amino acid sequence as depicted in SEQ ID No. 12 or a peptidomimetic generated from said fragment is provided. In another embodiment, a nucleic acid molecule encoding the isolated LRRC37B protein fragment is provided. Preferably, a host cell is also provided comprising the nucleic acid sequence, more particularly, heterologously expressing the nucleic acid sequence or with recombinant protein application. A non-limiting example of a host cell is a HEK cell.
SEQ ID No. 9 (cDNA SCN1B)
ATGGGGAGGCTGCTGGCCTTAGTGGTCGGCGCGGCACTGGTGTCCTCAGCCTGCGGGGGCTGCGTGGAGGTGG ACTCGGAGACCGAGGCCGTGTATGGGATGACCTTCAAAATTCTTTGCATCTCCTGCAAGCGCCGCAGCGAGACC AACGCTGAGACCTTCACCGAGTGGACCTTCCGCCAGAAGGGCACTGAGGAGTTTGTCAAGATCCTGCGCTATGA GAATGAGGTGTTGCAGCTGGAGGAGGATGAGCGCTTCGAGGGCCGCGTGGTGTGGAATGGCAGCCGGGGCAC CAAAGACCTGCAGGATCTGTCTATCTTCATCACCAATGTCACCTACAACCACTCGGGCGACTACGAGTGCCACGT CTACCGCCTGCTCTTCTTCGAAAACTACGAGCACAACACCAGCGTCGTCAAGAAGATCCACATTGAGGTAGTGGA CAAAGCCAACAGAGACATGGCATCCATCGTGTCTGAGATCATGATGTATGTGCTCATTGTGGTGTTGACCATATG GCTCGTGGCAGAGATGATTTACTGCTACAAGAAGATCGCTGCCGCCACGGAGACTGCTGCACAGGAGAATGCCT CGGAATACCTGGCCATCACCTCTGAAAGCAAAGAGAACTGCACGGGCGTCCAGGTGGCCGAA
SEQ ID No. 10 (amino acid sequence SCN1B)
MGRLLALVVGAALVSSACGGCVEVDSETEAVYGMTFKILCISCKRRSETNAETFTEWTFRQKGTEEFVKILRYENEVLQ LEEDERFEGRVVWNGSRGTKDLQDLSIFITNVTYNHSGDYECHVYRLLFFENYEHNTSVVKKIHIEVVDKANRDMASIV SEIMMYVLIVVLTIWLVAEM IYCYKKIAAATETAAQENASEYLAITSESKENCTGVQVAE
SEQ ID No. 11 (cDNA from LRRC37B specific domain)
GAGGTGGTTCCGCTTCTCAACCGGGATCAGAACCAGGCCCTAGTTCAGCTTCCTCGCCTCAAGTGGGTTCAAACT ACAGATCTAGATCGGGCTGCAGGTCATCAGGCAGATGAAATACTTGTTCCACTAGACAGTAAGGTTTCAAGACC AACCAAATTTGTT
SEQ ID No. 12 (amino acid sequence from LRRC37B specific domain)
EVVPLLNRDQNQALVQLPRLKWVQTTDLDRAAGHQADEILVPLDSKVSRPTKFV
In one embodiment, the application provides an LRRC37B peptide or protein fragment of at most 100 amino acids, at most 90, at most 80, at most 70 or at most 60 amino acids comprising the amino acid sequence as depicted in SEQ ID No. 12. SEQ ID No. 12 depicts a 54 amino acids long sequence comprising an LRRC37B specific domain, referred to herein as the "LRRC37B specific fragment" or "B specific fragment" or "LRRC37B specific domain" or "B specific domain". In a particular embodiment, the application provides an LRRC37B protein fragment consisting of SEQ ID No. 12. Also a homologue is provided of the LRRC37B specific fragment comprising at least 90%, 91%, 92%; 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the LRRC37B specific fragment as depicted in SEQ ID No. 12, wherein the homologue has the same or similar function as the LRRC37B specific fragment as depicted in SEQ ID No. 12. In another embodiment, an LRRC37B protein fragment is provided consisting of an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to SEQ ID No. 12, wherein the LRRC37B protein fragment still has the same or similar activity compared to SEQ ID No. 12. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.
In one embodiment, the amino acid sequence depicted in SEQ ID No. 12 or the homologue thereof refers to an isolated protein or peptide. According to another embodiment, the amino acid sequence depicted in SEQ ID No. 12 or the homologue thereof is generated by chemical amino acid synthesis. According to another embodiment SEQ ID No. 12 or the homologue thereof is generated by recombinant production.
Also provided is a molecule comprising the LRRC37B specific fragment as depicted in SEQ ID No. 12 respectively, or a homologue thereof and an additional entity. Said entity can be a biological, chemical or another protein or protein fragment. In one embodiment, said entity is a half-life extension entity and/or an entity that facilitates the molecule to cross the blood brain barrier. In particular embodiments, said molecule is a chimeric molecule, a chimeric protein, a dimeric protein, a fusion protein, a composition, a combination, a peptide or a polypeptide.
Also provided is a fusion protein or a pharmaceutical composition comprising an LRRC37B protein fragment, wherein the LRRC37B protein fragment is the LRRC37B specific fragment as depicted in SEQ ID No. 12 or any of the homologues thereof as described herein.
In a particular embodiment, a peptidomimetic of the LRRC37B specific fragment is provided. In one embodiment, the peptidomimetic comprises at least one D-amino acid and/or at least one non-natural amino acid.
Also provided is a pharmaceutical composition comprising any of the above LRRC37B protein fragments or homologue or peptidomimetics thereof.
The application also provides a nucleic acid molecule encoding the LRRC37B specific fragment or homologue thereof as described above, more particularly a nucleic acid molecule as depicted in SEQ ID No. 11. In another embodiment, a vector is provided comprising the nucleic acid molecule encoding the LRRC37B specific fragment as depicted in SEQ ID No. 12 or as depicted in SEQ ID No. 11.
A host cell is also provided comprising the nucleic acid molecule or vector, more particularly heterologously expressing the nucleic acid sequence or with recombinant protein application. A nonlimiting example of a host cell is a HEK cell.
Any of the LRRC37B protein fragments comprising the B specific domain or homologues thereof as described herein or nucleic acid molecules encoding them or vectors comprising the nucleic acid molecules are provided for use as a medicine, more particularly for use to modulate neuron excitability, even more particularly for use to decrease neuron excitability, even more particularly for use to treat seizures, epilepsy or epileptogenesis.
Also any of the peptidomimetics or fusion proteins or pharmaceutical compositions disclosed herein is provided for use as a medicine, more particularly for use to modulate neuron excitability, even more particularly for use to decrease neuron excitability, even more particularly for use to treat seizures, epilepsy or epileptogenesis.
Screening methods to identify modulators of LRRC37B-SCN1B
In a fifth aspect, screening methods are provided to identify modulators of the LRRC37B-SCN1B interaction or alternatively phrased to identify modulators of neuronal excitability.
In one embodiment, the modulator increases or reduces the LRRC37B-SCN1B interaction. The application thus provides a method, more particularly an in vitro method, of identifying a modulator of neuronal excitability or a modulator of the LRRC37B-SCN1B interaction, said method comprises the steps of: i) providing LRRC37B, or a fragment thereof comprising or consisting of the B-specific domain as depicted in SEQ ID No. 12; ii) providing SCN1B protein as depicted in SEQ ID No. 10 or a fragment thereof; iii) providing a test compound that is candidate for being a modulator of neuronal excitability or of the LRRC37B-SCN1B interaction or providing a peptidomimetic derived from the B-specific domain as depicted in SEQ ID No. 12; iv) contacting LRRC37B or fragment thereof provided in i) with the SCN1B protein or fragment thereof provided in ii) in the presence or absence of the test compound or of the peptidomimetic provided in iii); v) identifying as modulator of neuronal excitability or of the LRRC37B-SCN1B interaction, from iv) the test compound or peptidomimetic that is, compared to identical conditions but for the absence of the compound, statistically significantly reducing or increasing the binding of the SCN1B protein or fragment thereof to LRRC37B or the fragment thereof.
In a particular embodiment, the binding of LRRC37B or the fragment thereof to the SCN1B protein or the fragment thereof is determined by immunologic, fluorescent or radiologic detection, co-sedimentation, co-immunoprecipitation, or electron microscopy. In an embodiment, any other method to determine the binding known in the prior art can be used. Assays for studying protein-protein interactions are well- known by the person skilled in the art. A non-limiting example is the fluorescence polarization (FP) assay. In another particular embodiment, the LRRC37B or the fragment thereof in step i) is provided by one or more cells expressing LRRC37B or the fragment thereof.
In a preferred embodiment, a method of identifying a modulator of neuron excitability is provided, said method comprising the steps of: generating a peptide consisting of less than 30 contiguous amino acids wherein the peptide is a fragment of a LRRC37 B-specific domain as depicted in SEQ ID No. 12 or generating a peptidomimetic of said peptide; providing a SCN1B protein as depicted in SEQ ID No. 10 or a functional fragment thereof; contacting the SCN1B protein with the peptide or peptidomimetic or with the LRRC37 B- specific domain as depicted in SEQ ID No. 12; identifying as a modulator of neuron excitability a peptide or peptidomimetic that, compared to identical conditions shows a statistically significantly increased binding to SCN1B compared to the LRRC37 B-specific domain as depicted in SEQ ID No. 12.
By identical conditions, as referred to herein, it is meant that the binding of the SCN1B protein with the peptide or peptidomimetic is done under the same conditions, and using the same parameters, such as concentration, assay, temperature, etc. as the conditions in which the binding of the LRRC37 B-specific domain as depicted in SEQ ID No. 12 to SCN1B compared was determined.
In an embodiment, a method of identifying a modulator of neuron excitability is provided comprising the steps of: i) generating a plurality of peptides comprising 30, 25, 20, 15 or 10 or less contiguous amino acids wherein the peptides are fragments of the LRRC37 B-specific domain as depicted in SEQ ID No. 12 or generating a plurality of peptidomimetics from said peptides; ii) providing the SCN1B protein as depicted in SEQ ID No. 10 or a functional fragment thereof; iii) contacting the SCN1B protein provided in ii) with one or more of the peptides or peptidomimetics provided in step i); iv) identifying as a modulator of neuron excitability from iii) a peptide or peptidomimetic that, compared to identical conditions shows a statistically significantly increased binding to SCN1B compared to the LRRC37 B-specific domain.
More particularly, the method comprises an additional step of providing the NAVI.6 protein as depicted in SEQ ID No. 14 or a fragment thereof that binds SCN1B, wherein the NAVI.6 protein or fragment thereof is contacted with the SCN1B and wherein one or more of the LRRC37B peptides or peptidomimetics are added to the protein complex. The one or more peptides or peptidomimetics are then identified as a modulator of neuron excitability when the peptide or peptidomimetic statistically significantly reduces the binding between SCN1B and the NAVI.6 protein or fragment thereof.
Current application also provides methods for designing, selecting, screening and/or optimizing a chemical entity that binds to and/or modulate the activity of the NAVI.6 channel, said methods comprise the steps of: a. generating a co-crystal comprising the SCN1B protein and the LRRC37B specific fragment as depicted in SEQ ID No. 12 or a co-crystal comprising the SCN1B protein and the NAVI.6 protein as depicted in SEQ ID No. 14 or fragment thereof that binds SCN1B; b. determining the three-dimensional atomic coordinates of the co-crystal by X-ray diffraction; c. providing the three-dimensional atomic coordinates of the co-crystal on a computer comprising the means for generating three-dimensional structural information from atomic coordinates; and b. designing, selecting, screening and/or optimizing the chemical entity by performing a fitting operation between the chemical entity and the three-dimensional structural information of all or part of said crystal.
EXAMPLES
Example 1. LRRC37 gene family is selectively expressed in human cortical neurons
The LRRC37 (Leucine-Rich Repeat-containing protein 37) gene family encodes putative orphan receptors of unknown function. The number of LRRC37 genes increased in simian species leading to 3 encoding paralogs in the chimpanzee and 4 in the human genomes, compared to two in the mouse genome. LRRC37 genes encode A and B types of transmembrane proteins comprising an LRR in their extracellular part. The B type LRRC37 carries a specific domain, hereafter referred to as B domain or the LRRC37B specific domain, located near the N-terminal part of the protein. The LRRC37B type emerged in the simian genomes. At the transcriptome level, no LRRC37 transcript is detected in the mouse cortex throughout life, while all human paralogs are detected in the human cortex, with lower levels in the macaque cortex (Figure 1A). It is expressed in a subset of all cortical neurons whose proportion increases throughout life to reach 30-50% of pyramidal neurons at adulthood (Figure 1B-C) while the absolute amount in cortical neurons are stable through life (Figure IE). It is enriched in human cortical neurons compared to chimpanzee (Figure ID). LRRC37B type is thus uniquely expressed in simians, and LRRC37B is expressed at a higher levels in the human cerebral cortex.
We next determined LRRC37B at the protein level (uniprot ID: Q96QE4) in the adult human cerebral cortex, using a specific antibody that only recognizes the B and not the A types. Remarkably this revealed a selective localization of LRRC37B at the level of the axon initial segment (AIS - as determined with AIS marker Ankyrin G) of a fraction of pyramidal neurons (Figure 2A-B). Time-course analysis at several stages from neonatal to >60 years old specimens, revealed that the proportion of neurons sharply increased from birth until childhood to stabilize at puberty (Figure 2F). Importantly, similar stainings on adult cerebral cortex of mouse, macaque and chimpanzee with the same antibody did not reveal any detection at the AIS, strongly suggesting that LRRC37B is a human cortical neuron-specific protein located at the axon initial segment.
Example 2. LRRC37B negatively regulates cortical pyramidal neuron excitability
In order to probe the function of LRRC37B, we first performed gain-of-function in mouse pyramidal neurons through sparse in utero electroporation in the cortex (at embryonic day 15.5, thereby targeting mostly cortical layer 2-3 pyramidal neurons), followed by analysis at P21. We then detected the LRRC37B protein enriched at the axon initial segment (AIS), colocalized with ankyrin-G (Figure 2C). We detected no differences in the AIS length and location compared with control neurons.
The AIS is a subcellular compartment critical for neuronal excitability, as the main site of generation of action potentials (AP) through the opening of sodium-voltage gated channel a subunits (NAVa) concentrated at this site (Leterrier 2016 Curr Top Membr 77, 185-233). We analyzed control and LRRC37B-expressing neurons using patch-clamp electrophysiological recordings in slices of electroporated animals P24-P32. LRRC37B gain-of-function led to a striking decrease in neuronal excitability, characterized by a lower AP firing rate in current clamp (Figure 2D(a)), a higher rheobase and an increased AP risetime and AP width (Figure 2E). The AIS is specifically targeted by chandelier GABA-ergic interneurons that thereby regulate pyramidal cell output (Gallo et al 2020 Trends Neurosci 43, 565-580). We therefore investigated the synaptic properties of mouse cortical neurons following LRRC37B gain-of-function. However, patch-clamp recordings detected no differences in excitatory and inhibitory postsynaptic potentials and quantification of GABA-ergic synapses at the level of AIS detected no differences either.
These data indicate that LRRC37B acts as a negative regulator of neuronal excitability, mostly at an intrinsic level.
Example 3. LRRC37B binds to FGF13A ligand, and thereby negatively regulates voltage gated channels
To understand the molecular mechanisms of action of LRRC37B, we searched for binding partners of LRRC37B. First, an ELISA-like binding assays was performed using as a bait LRRC37B fused with alkaline phosphatase, which was applied to 920 transmembrane or secreted proteins as partner candidates, including 26 proteins enriched at the AIS or in chandelier interneurons. This led to a single reproducible hit and thus potential ligand to LRRC37B, namely FGF13 isoform 1 or FGF13A (uniport ID: Q92913-1). This is a rather surprising result as FGF13 was previously described as a member of the FHF non-canonical FGF family, which is thought to encode non-secreted proteins (Dover et al 2010 J Physiol 588, 3695-3711; Smallwood et al 1996 Proc Natl Acad Sci USA 93, 9850-9857; Wang et al 2012 Structure 20, 1167-1176). FGF13 encodes several splicing isoforms (FGF13A, FGF13B, FGF13V, FGF13Y and FGF13VY) that share C terminal exons (regions 2-5 as visualized in Figure 3A upper panel, "core domain") and differ by their initial first exon (Munoz-Sanjuan et al 2000 J Biol Chem 275, 2589-2597) (Figure 3A). Using heterologous expression in HEK cells, we first found that FGF13A, and not the other isoforms, is selectively secreted by the cells, thus potentially enabling it to reach the extracellular compartment to bind to LRRC37B (Figure 3A lower panel). We next tested the interaction of the FGF13 isoforms with LRRC37B coexpressed in heterologously transfected HEK-293 cells. LRRC37B was found to be coimmunoprecipitated only with FGF13A and not with any of the other isoforms (Figure 3B). This specific interaction was confirmed by adding a recombinant protein corresponding to FGF13A to the culture medium of cells expressing LRRC37B, which was found to be co-immunoprecipitated with LRRC37B (Figure 3C). Similar binding results were obtained with a smaller synthetic peptide corresponding only to the first exon of FGF13A (ExonS, Figure 3C).
Collectively these data indicate that FGF13A is an extracellular ligand of LRRC37B, to which it binds to through its N-terminal domain.
To identify the domains in the LRRC37B receptor responsible for the interaction with FGF13A, we tested binding properties of deletion constructs expressing complete or parts of the extracellular part of LRRC37B fused with the transmembrane domain of PDGF-R (Figure 3D-E). We performed immunoprecipitations of the different LRRC37B constructs co-transfected with FGF13A in HEK-293 cells. This revealed that the LRR domain is necessary and sufficient to bind to FGF13A (Figure 3D).
LRRC37B thus constitutes a selective receptor for FGF13A, to which it binds to through its LRR domain.
Example 3(b). Determining the minimal binding fragment of FGF13A S-domain (minimal binding fragment of "Exon S")
To identify the binding site of FGF13A, more particularly FGF13A S-domain or S-fragment or ExonS, to LRRC37B receptor responsible for the interaction with FGF13A, we tested the direct interaction between a recombinant protein corresponding to the LRR of LRRC37B and a membrane with distinct synthetized peptides (10 amino acid length) covering the FGF13A S-domain (herein below also referred to as "ExonS") sequence shown in Figure 3F. It revealed 2 potential binding sites: amino acids 4-23 and 40-63. Then, with performed affinity measures using fluorescent polarization assay between recombinant protein LRR and synthetic peptides fused to a fluorescent protein. This confirmed the direct binding of the ExonS to LRR (affinity of 530 nM) and of "ExonS 40-63" (affinity of 160nM), which ExonS 40-63 corresponds to a peptide of a SEQ. ID No. 15, but not of negative controls (ExonU of FGF13B isoform, random peptides) (Figure 3G). Binding properties were also assessed by applying synthetic peptides fused to biotin to live HEK-293T cells transfected for the extracellular domain of LRRC37B (versus an empty vector): they showed a binding of ExonS 40-63 (affinity of 0.4nM) and ExonS (affinity of 4nM) but not ExonS 4-23 (Figure 3H), said ExonS 4-23 corresponding to a peptide of SEQ. ID No. 16. To conclude, this shows that LRRC37B binds to the ExonS 40-63 amino acids of FGF13A.
Example 4. Extracellular FGF13A decreases neuronal excitability by binding to NAV channels, which is enhanced by LRRC37B
We next tested for potential effects of FGF13A on the excitability of mouse pyramidal neurons. To this aim we performed patch-clamp recordings of mouse cortical neurons combined with the extracellular addition of FGF13A (Figure 4A(a) and (b)). This revealed a dose-dependent effect of FGF13A on neuronal excitability, leading to decreased AP firing rate, increased rheobase, as well as a decrease in ionic currents (Figure 4B(a)-(b), D-E). Similar results are obtained with the S-fragment (encoded by ExonS) extracellular application (Figure 4C, left, Figure 4F-G), and the ExonS 40-63 extracellular application (Figure 4A(b),4B(b),4D(b),DE(b)). Importantly, similar experiments performed with intracellular application of FGF13A led to no obvious effects on neuronal excitability (Figure 4C, right, Figure 4C(b)), indicating that it acts mostly or exclusively extracellularly on neuronal excitability. This is in contrast with previous reports that used much higher (mM) concentrations of FGF13A (Barbosa et al 2017 Pflugers Arch Eur J Physiol 469, 195-212; Dover et al 2010 J Physiol 588, 3695-3711).
Based on these results, we tested whether FGF13A could bind to the voltage-gated sodium channel a subunit NAVI.6, the predominant effector of neuronal excitability at the AIS of cortical neurons. Interestingly, using heterologous expression in HEK-293 cells, we found that FGF13A immunoprecipitates with NAVI.6 even in the absence of LRRC37B (Figure 4H). Conversely LRRC37B alone could not be immunoprecipitated with NAVI.6, but could be if co-transfected with FGF13A. These data thus indicate that FGF13A binds directly to NAVI.6 and to LRRC37B, and that the three proteins can be found in the same complex when co-expressed together.
The above findings could be confirmed in vivo. First, co-immunoprecipitation experiments in mouse cortex samples expressing LRRC37B-HA (following in utero electroporation (IUE)) revealed binding between FGF13A and LRRC37B in vivo (Figure 41). Second, immunostainings in the mouse cortex overexpressing LRRC37B confirmed the localization of FGF13A at the AIS and co-localisation of LRRC37B, FGF13A and NAVI.6 (Figure 4J). We performed co-occurrence measures on the STED pictures (Figure 4J) and extracted the Pearson correlation coefficient to assess pixel-to-pixel colocalization of (1) LRRC37B with FGF13A immunoreactivity, and (2) LRRC37B and pan-NAVa immunoreactivity. These data and analyses indicate a high co-occurrence between LRRC37B and NAVa (r=0.57 ± 0.17) and moderate cooccurrence between LRRC37B and FGF13A (r=0.32 ± 0.17). Interestingly, following expression of LRRC37B, FGF13A was found to be more abundant at the AIS of mouse neurons in distinctive patches (Figure 4K-L), while the abundance of NAV a subunits appeared to be unchanged (Figure 4M). Third, we can co-immunoprecipitate NAVa subunits (among them NAVI.6), LRRC37B and FGF13A from human cerebral cortex (Figure 6A). Interestingly, when comparing the molecular properties of human LRRC37B with macaque and chimpanzee orthologous proteins we found that while the chimpanzee protein displayed the same binding properties to FGF13A, the macaque protein did not display binding to FGF13A, in line with divergence in LRR domain organization.
These data suggest a model whereby LRRC37B can concentrate FGF13A at the AIS, thereby enhancing its inhibitory effect on NAV a subunits, leading to decreased neuronal excitability.
Example 5. LRRC37B binds to NAV beta subunit 1 (SCN1B) of the voltage-gated channel through its B- specific domain
As a second approach to identify binding partners of LRRC37B, we performed ex vivo affinity purification of rat brain extracts using the extracellular domain of LRRC37B as a bait. This revealed several AIS potential partners, including NAV a (SCN1A, SCN2A) and their regulatory NAV beta subunit SCN1B. The interaction was confirmed using heterologous co-expression in HEK cells, in which SCN1B, LRRC37B, and FGF13A could be found in the same immunoprecipitated complex (Figure 5A). Using deletion mutants (Figure 5B), we found that the B domain, found only in LRRC37B and not in other paralogs, was necessary and sufficient to mediate the binding between LRRC37B and SCN1B (Figure 5C). Moreover, we found that LRRC37B could inhibit the binding of SCN1B to NAVI.6, suggesting competitive interactions between LRRC37B, SCN1B and NAVI.6 (Figure 5D). Interestingly, these effects could be mimicked by a synthetic LRRC37B protein fragment (LB133-186) encoding the B domain only, which was also found to bind directly to SCN1B (Figure 5E) and thereby inhibit its binding to NAVI.6 (Figure 5F). Consistent with the importance of the B domain for SCN1B interaction, we detected no interaction between LRRC37A receptors of human and non-human species, which lack this domain, and SCN1B. To summarize, these data indicate that LRRC37B can directly bind to SCN1B and influence its interaction with NAVI.6, which could constitute an additional mechanism of NAV regulation.
Example 6. LRRC37B regulates human cortical neuron excitability.
Next, LRRC37B, FGF13A, SCN1B and SCN8A (NAVI.6) were co-expressed in human cortical neurons. Coimmunoprecipitation experiments from human cortex tissue confirmed that LRRC37B, SCN1B, and FGF13A are present in the same complex (Figure 6A). Then we tested the physiological relevance of this complex by performing acute patch-clamp recordings on human ex vivo temporal cortex biopsies. We focused on L2/3 neurons, for which only a subset of cells expresses LRRC37B (Figure 1B-C), and performed post-hoc LRRC37B immunostainings to compare the properties of LRRC37B-positive vs LRRC37B-negative neurons. This revealed that LRRC37B+ neurons display less excitability than LRRC37B- neurons, with a decreased AP firing rate (Figure 6B(a), 6B(b)), an increased rheobase (Figure 6C) and AP risetime (Figure 6D) and width (Figure 6C-E).
Overall these data indicate that LRRC37B interacts with FGF13A and a voltage-gated channel complex in the human cortex, thereby regulating neuronal intrinsic excitability.
Materials and Methods
Genome and transcriptome analysis. Encoding genes paralogs and orthologs originated from (Giannuzzi et al., 2012) and Ensembl gene trees (https://www.ensembl.org). Transcriptomic comparison between species is an analysis of data from Henrik Kaessmann laboratory (Heidelberg, Germany) described in (Cardoso-Moreira et al., 2020) and available at https://apps.kaessmannlab.org/evodevoapp/. Genes considered are the following: ENSG00000176681(human LRRC37A), ENSG00000238083 (human LRRC37A2), ENSG00000176809 (human LRRC37A3), ENSG00000185158 (human LRRC37B), ENSMMUG00000008199 (macaque LRRC37-M1), ENSMMUG00000063877/ ENSMMUG00000011880 (macaque LRRC37-M2), ENSMMUG00000018463 (macaque LRRC37-M7), ENSMUSG00000078632 (mouse LRRC37A) and ENSMUSG00000034239 (mouse GM884). Transcriptomic expression in the human cerebral cortex has been analysed from data available at the UCSC cell browser (autism data) taking control individuals available at https://cells.ucsc.edu/?ds=autism and published in (Velmeshev et al.,
2019) and at Ml - 10X GENOMICS (2020) Allen Brain Single Cell Atlas (https://portal. brainmap. org/atlases-and-data/rnaseq) and published in (Bakken et al., 2021). Copy number estimates for genes LRRC37A, LRRC37A2, LRRC37A3, and LRRC37B were obtained using QuicK-mer2 (Shen & Kidd,
2020) in windows of 500 unique kmers. High-coverage whole-genome sequencing data form 2,504 unrelated individuals from five continental "super populations" (Byrska-Bishop et al., 2022) were downloaded in cram format and used as input for QuicK-mer2 with T2T-CHM13 (vl.O) as reference (Nurk et al., 2022). We genotyped overall gene CN as the mean CN across the gene body using a custom python script. CN-dotplots generated using the R package ggplot2.
Human and non-human primate cortex immunostaining. For immune fluorescent staining, human vibratome and cryosections as well as nonhuman primate sections were stained using Cy3 TSA amplification for LRRC37B. Briefly, slice were treated with tap water (5mn for vibratome sections, lmn for cryosections), BLOXXAL reagent (3 hours for vibratome sections, lOmn for cryosections), three TNT washes (0.1 M TRIS-HCI, pH 7.5, 0.15 M NaCI, 0.3% Triton), TNB incubation for 2 hours and then incubation in TNB with rabbit anti-LRRC37B 1:1000 antibody which recognizes the LB specific domain (HPA015135, Merck) at 4°C (overnight for cryosections, 3 days for vibratome sections). After five washes with TNT, sections were incubated overnight at 4°C with anti-rabbit IgG antibody conjugated with HRP 1:100. Cy3 TSA reaction was performed after five washed with TNT (lOmn reaction for vibratome sections, 3mn for cryosections). Slices were then transferred into the blocking solution (PBS 0.3% Triton, 5% horse serum, 3% BSA) and incubated for 1 hour. Brain slices were if required incubated at 4°C with mouse anti-ankyrin-G (1:500; MABN466, Merck) antibody (3 days for vibratome sections, overnight for cryosections). After three PBS washes, slices were incubated overnight at 4°C with donkey anti-mouse a488 or a647 and Hoechst (1:10000). For patched sections, brain slices were directly incubated overnight in PBS at 4°C containing streptavidin-a488 1:500 and Hoechst 1:10000. After three washes in PBS, brain sections were mounted on a slide glass with the mounting reagent (DAKO glycerol mounting medium) using #1.5 coverslips.
DNA constructs. LRRC37B cDNA originates from IRCMp5012D0514D (SourceBiosciences) whose sequence miss ExonS 4-5 which have been amplified by PCR from a cDNA library derived from GW18 fetal cortex (Suzuki et al., 2018) using the primers designed on the basis of the sequence of reference genome. The size of PCR fragment was confirmed and PCR fragment was subcloned into the Bsmbl and Ecorl restriction sites of the original cDNA by In Fusion cloning. LRRC37B cDNA has been inserted by PCR amplification and InFusion cloning into the multicloning site between CAG promotor and IRES in the lentiviral backbone pCIG (CAG-IRES-EGFP-WPRE, Addgene #122953) (Suzuki et al., 2018) and pCIG-LSL (CAG-LSL-IRES-EGFP-WPRE) described in (Iwata et al., 2020). Resulting pCIG-LRRC37B and pCIG-LSL- LRRC37B plasmids have been used compared to pCIG and p-CIG-LSL in in utero electroporation experiments. LRRC37B cDNA has been PCR amplified adding a Cter HA tag and inserted into the pCIG backbone at the multicloning site between CAG promotor and IRES. Resulting pCIG-LRRC37B-HA plasmid has been used compared to pCIG in in utero electroporation experiments. All constructs were verified by DNA sequencing. Predicted extracellular sequence lacking the signal peptide of LRRC37B (LRRC37BECTO) corresponding cDNA has been cloned by PCR amplification into a modified pCMV6-XL4 as described in (Apostolo et al., 2020) leading to pLRRC37B-Fc using InFusion cloning. Fc-fusion protein contain a prolactin leader peptide (PLP) followed by an N-terminal FLAG tag, ectodomain of interest, a 3CPro cleavage site, and the dimeric human Fc domain. Similarly, LRRC37B-alkaline phosphatase (AP) fusion protein contain a leader peptide as described in (Apostolo et al., 2020), a FLAG tag and ectodomain of interest. All constructs were verified by DNA sequencing. Predicted extracellular sequence lacking the signal peptide of LRRC37B (LRRC37Becto) corresponding cDNA and truncated versions have been cloned by PCR amplification into pDisplay™ Mammalian Expression Vector (ThermoFisher V66020) using InFusion cloning resulting in pHA-LRRC37B-ECTO (amino acids 28 - 905), pHA-LRRC37B-ALRR-ECTO (amino acids 28 - 522: 748 - 905), pHALRRC37B-LRR-ECTO (amino acids 468 - 841), pHA-LRRC37BALB-ECTO (amino acids 186 - 905), pHA-LRRC37B-LB-ECTO (amino acids 28 - 520). All constructs were verified by DNA sequencing. FGF13A cDNA plasmid originates from Origene (RC204164) and has been PCR amplified for insertion into pCIG plasmid by InFusion cloning. FGF13B, FGF13VY, FGF13core have been PCR amplified from FGF13A cDNA with primers targeting the core domain of FGF13 and with ExonS B or VY in the 5' primer. All constructs were verified by DNA sequencing. pCMV-macaqueLRRC37B-HA (ORF XP_028692824.1) and pCMVchimpanzeeLRRC37B-HA (ORF CK820_G0028539) have been synthetized by GenScript and inserted in pcDNA3.1(+)-C-HA backbone (Addgene #128034). All constructs were verified by DNA sequencing. pCMV-SCNIB-FLAG, plasmid originates from Origene (RC209565, RC215868, RC219002). Predicted extracellular sequence lacking the signal peptide of SCN1B (SCNIBecto) corresponding cDNA has been cloned by PCR amplification into pDisplay™ Mammalian Expression Vector (ThermoFisher V66020) using InFusion cloning resulting in pHA-SCNIBecto. All constructs were verified by DNA sequencing. pCMV-SCN8A-IRES-Scarlet plasmid originates from Addgene (#162280) and has been described in (DeKeyser et al., 2021). pCAG-cre is a gift from Franck Polleux laboratory (United States) described in (Hand et al., 2005). pCAG-GEPH.FingRtdTomato-IL2RGTC is a gift from Juan Burrone (United Kingdom), derived from pCAG_GPHN.FingR-mKate2-IL2RGTC (Addgene #46297) described in (Gross et al., 2013). pGPR158_ECTO, pSLIRTK2_ECTO and pLRRTMl_ECTO were previously described (Condomitti et al., 2018; Schroeder et al., 2018). pCD4_ECTO is a gift from Lui's Ribeiro (Joris de Wit's laboratory), with the cDNA of the predicted extracellular domain of CD4, originating from pCMV-CD4 (Addgene #51604) described in (Raissi et al., 2013), inserted into the pDisplay™ Mammalian Expression Vector (ThermoFisher V66020).
Live staining of HEK-293T cells. HEK-293T cells were split on coverslips and have been transfected with 500ng of each construct total amount (lug) of DNA (pCIG + pCIG-humanLRRC37B-HA or + pCMVchimpanzeeLRRC37B-HA or + pCMV-macaqueLRRC37B-HA or + pCIGhumanLRRC37A2-HA), using XtremeGene9 transfection reagent. For live staining, 48 hours after transfection, medium was washed with cold PBS and then rabbit anti-LRRC37B (1:1000, as described above) was applied in PBS for lh at 4°C. This was followed by washes in PBS, fixation 30mn with PBS PFA 4% at 4°C. After washes in PBS, cells were blocked for lh in PBS 5% HS 3% BSA, and then with 1:1000 donkey anti-rabbit Cy3 and Hoechst 1:10000 for 2 hours in the same solution at room temperature. For staining after fixation and permeabilization, cells were washed with cold PBS and fixed 30mn with PBS PFA 4% at 4°C. After washes in PBS, cells were blocked for lh in PBS 5% HS 3% BSA, and then with rabbit anti-HA (1:1000; CST 3724S, Bioke) overnight at 4°C in the same solution. After washes in PBS, 1:1000 donkey anti-rabbit Cy3 and Hoechst was applied for 2 hours in the same blocking solution at room temperature. Coverslips were mounted on a slide glass with the mounting reagent (DAKO glycerol mounting medium).
In utero electroporation. Barrel cortex of E15.5-day-old embryos of timed-pregnant CD1 mice were unilaterally electroporated with lentiviral plasmids. Briefly, the dam was anesthetized with isoflurane following buprenorphine and carprofen injection, and the uterus exposed. A solution of 1-2 pg/pl DNA and 0.01% fast green dye was injected into the embryonic lateral ventricle with a heat-pulled glass capillary. For immunostainings and image analysis, pCIG-LSL plasmids (1000 ng/pL) were used together with pCAG-cre (15 ng/ pL) eventually with pCAG-GEPH.FingR-tdTomato-IL2RGTC while for electrophysiology pCIG plasmids were used (1 pg/pl) (see above). The embryo's head was then placed between the paddles of pair of tweezer electrodes with the cathode lateral to the filled ventricle and five 50 ms, 30 V pulses were delivered with an interval of 950ms by a BTX830 electroporator (Harvard Apparatus). After electroporation, the uterus was replaced, the incision sutured closed and placed on a heating plate until recovery.
Mouse cortex processing and immunostaining. Mouse P28 animals were perfused transcardiacally with ice-cold sucrose 8% PFA 4%. Brains were dissected and soaked in the same fixative for 3 hours, then stored in PBS azide. Then they either have been sectioned in 80 pm thickness using vibratome or 50 pm thickness using cryostat after dehydration in sucrose 30% and freezing in OCT. Slices were transferred into the blocking solution (PBS 0.3% Triton, 5% horse serum, 3% BSA) and incubated for 1 hour. Brain floating slices were incubated 3 days at 4°C with primary antibodies: rabbit anti-LRRC37B (1:1000, as described above), mouse anti-ankyrin-G (1:500, as described above), mouse anti-FGF13A (1:500; MAS- 27705, ThermoFisher), chicken anti-EGFP (1:1000; abl3970, Abeam), rat anti-mCherry which recognizes tdTomato (1:1000; M11217, ThermoFisher), mouse anti-pan-NAVa (1:500; S8809, Merck). For VGAT staining (guinea pig anti-VGAt 1:500; 131 004, Synaptic Systems), stainings have been done sequentially in blocking solution PBS 1% Triton, 5% horse serum, 3% BSA. After three PBS washes, slices were incubated overnight at 4°C with secondary antibodies in PBS: donkey anti-rabbit Cy3, anti-rabbit a594, antimouse a647, anti-chicken a488, anti-rat Cy3, anti-guinea pig a647 (1:1000 or 1:250 for cryosections used for STED imaging) and Hoechst (1:10000). After three washes in PBS, brain sections were mounted on a slide glass with the mounting reagent (DAKO glycerol mounting medium) using #1.5 coverslips.
Image acquisition. Confocal images were obtained with Zeiss LSM880 and LSM900 driven by Zen Black and Blue software equipped with objectives lOx, 20x, oil immersion 25x and oil immersion 40x, AiryScan system and argon, helium-neon and 405 nm diode lasers. STED single focal section images were obtained with an Abberior system with Olympus 1X83 body equipped with lOOx oil immersion, 480, 532, 640 nm excitation lasers and 595nm 775 nm depletion lasers. STED pictures were deconvoluted using Huygens deconvolution software. Except if specified, representative pictures are maximum projections.
Image analysis. AIS intensity profile was done on maximum projection pictures in Matlab as described in (Grubb and Burrone, 2010). In mouse, the beginning of the AIS was set using the EGFP channel (starting from the soma). In human, ankyrin-G was used to set the beginning of the axon. Puncta and area quantification have been done using Fiji. On average, 10 focal sections (0.4 pm thickness) maximum projection was used for quantification. For STED imaging, one focal section was used only with on average 10pm length in the proximal part of the AIS. EGFP was used to delineated the soma and the AIS (30pm starting from the soma). Binarized pictures of the gephyrin-tdTomato, VGAT, FGF13A have been used to quantify manually the number of puncta or their positive area.
Electrophysiological recordings. For mouse experiments, coronal slices were prepared from postnatal day P24-32 animals. Briefly, after decapitation, the brain was quickly removed and transferred into ice- cold cutting solution (in mM): 87 NaCI, 2.5 KCI, 1.25 NaH2PO4, 10 glucose, 25 NaHCO3, 0.5 CaCI2, 7 MgCI2, 75 sucrose, 1 kynurenic acid, 5 ascorbic acid, 3 pyruvic acid, pH 7.4 with 5% CO2/95% 02, and whole brain coronal slices (250 pm) were cut using a vibratome (VT1200, Leica Biosystems). Afterward, slices were transferred to 32 °C cutting solution for 45 min to recover and finally maintained at room temperature until used for recordings. Human cortical samples were collected at the time of surgery, immerged in ice-cold ACSF (NaCI 126 mM, NaHCO3 26mM, D-glucose lOmM, MgSO4 6mM, KCL 3mM, CaCI2 ImM, NaH2PO4 ImM, 295-305mM, pH adjusted to 7.4, with 5% CO2/95% 02) and transferred immediately into the laboratory, with processing (slicing for electrophysiology or protein extraction) in an interval of 5-10 minutes. Slicing solution contained choline chloride HOmM, NaHCO3 26mM, Na- ascorbate 11.6 mM, Dglucose lOmM, MgCI27mM, Na-pyruvate 3.1 mM, KCI 2.5 mM, NaH2PO4 1.25mM, CaCI2 0.5 mM; 300-315 mOsm, pH adjusted to 7.4, with 5% CO2/95% 02 and was ice-cold. Recovery solution was the same than the ACSF used for the transfer from hospital. Slicing was performed with a vibrating blade microtome or using a comprestome, and 300-pm slices were incubated for around 30 min at 32 °C in ACSF. Slices were then stored at around 20 °C until use for electrophysiological recordings. For recordings, mouse and human brain slices were continuously perfused (32-34°C) in a submerged chamber (Warner Instruments) at a rate of 3-4 ml/min with (in mM): 127 NaCI, 2.5 KCI, 1.25 NaH2PO4, 25 NaHCO3, 1 MgCI2, 2 CaCI2, 25 glucose at pH 7.4 with 5% CO2/95% 02. Whole-cell patch-clamp recordings were done using borosilicate glass recording pipettes (resistance 3.5-5 MO, Sutter P-1000), using a double EPC-10 amplifier under control of Patchmaster v2 x 32 software (HEKA Elektronik, Lambrecht/Pfalz, Germany). The following internal medium was used (in mM): 135 K-Gluconate, 4 KCI, 10 HEPES, 4 Mg-ATP, 0.3 Na2-GTP, 10 Na2- phosphocreatine, 3 biocytin (pH 7.3). Cell intrinsic properties were recorded in current clamp, while sEPSCs and sIPSCs were recorded in voltage clamp at -70 mV and 0 mV, respectively. Currents were recorded at 20 Hz and low-pass filtered at 3 kHz when stored. The series resistance was compensated to 75-85%. Cell intrinsic properties were analyzed using Fitmaster (HEKA Elektronik, Lambrecht/Pfalz, Germany), spontaneous input was analyzed using Mini Analysis program (Synaptosoft). For protein/peptide bath application experiments we used consecutive repeats of recordings (control followed by recordings 3 to 16 minutes after application), 10 minutes application is plotted. For initial intracellular application experiments, 50 nM FGF13A were added to the intracellular pipette solution and standard approach procedures were used. Next, to minimize FGF13A exposure of the slice, a fluorescent dye was added to the intracellular medium (Alexa568, 5 pM) to visually regulate minimal outflow of intracellular solution from the pipette during the approach and cell-attached phase of recordings. In both experiments (no application versus application), recordings were started 3 minutes after establishing whole-cell configuration, to allow infusion of FGF13A into the cell. Phase plot analysis compares the rate of change (first derivative) in voltage during APs (y-axis) to the membrane voltage (x- axis). The first current step (1 sec) to initiate AP(s) was used for phase plot analysis. The AIS, somatic and repolarization voltages were determined as the absolute membrane potential measured at the respective peaks.
Elisa assay. An ELISA-based assay (Ozgul et al., 2019) was used to identify the interaction between ectodomain (as defined in the Uniprot database) of 920 cell surface or secreted proteins cloned in frame with an Fc domain against LRRC37B-ECTO-AP fusion as described in (Apostolo et al., 2020). Horseradish peroxidase (HRP) conjugated anti-Fc antibody develops a blue colour if the prey (FGF13A) remain bound to the bait (LRRC37B) after the washes. After the initial identification, the experiment has been repeated 3 times using triplicate wells. The library contained AIS proteins or proteins coded by genes enriched in chandelier interneurons as described in (Bakken et al., 2021; Favuzzi et al., 2019; Leterrier, 2016): ALCAM, CDH4, CDH6, CDH11, CNTNAP5, DPP10, FGF13 isoform 1 (FGF13A), FSTL5, ITGAV, ITGA6, LRRN1, LRRN2, NFASC, OLFM3, PCDH19, PCSK2, ROBO1, SGCD, SLITRK1, SLITRK5, TENM4, THSD7A, UNC5B. They were all negative except FGF13A.
Protein extraction and immunoprecipitations.HEK293T have been transfected with 500-2000ng of DNA using XtremeGene9 transfection reagent. 72 hours after transfection, cells were lysed in lysis buffer (50mM Tris-HCI, pH 7.5, 1% sodium deoxycholate, 0.1% Triton, proteinase inhibitors) on a wheel for one hour in a cold room. When required, FGF13A and its ExonS where applied in the culture medium 5 hours before protein extraction. EGFP positive area of P17 mouse cortex has been dissected using forceps in cold PBS of brains after cervical dislocation. Cortices have been homogenized in homogenization buffer (50mM Tris-HCI, pH 7.5, 1% sodium deoxycholate, proteinase inhibitor) using a Dounce homogenizer. After Triton addition (0.1% final concentration), samples have been rotated on a wheel for one hour in a cold room. Human cortices (from 14yo-48yo patients) have been homogenized in homogenization buffer (50mM Tris-HCI, pH 7.5, 1% sodium deoxycholate, proteinase inhibitor) using a Dounce homogenizer. After Triton addition (0.1% final concentration), samples have been rotated on a wheel for one hour in a cold room. After incubation, samples were centrifugated 25mn 16000g and lysates transferred in a new tube with addition of NaCI (final concentration 150 mM). Samples were incubated overnight on a wheel in a cold room with HA magnetic beads or protein A magnetic beads coupled with lug of mouse anti-pan-NAVa (as described above) or rabbit anti-FLAG (abll62, Abeam) or mouse anti- FGF13A (as described above) or mouse IgG (ThermoFisher) antibodies. Beads were washed 4 times with the washing solution (50mM Tris-HCI, pH 7.5, 1% sodium deoxycholate, 0.1% Triton, 150 mM) and one time with PBS. Subsequently, samples were eluted in 2x Laemmli buffer at 95°C. Medium was centrifugated 25mn 16000g and diluted to final lx Laemmli buffer at 95°C. The input (in lx Laemmli buffer), medium, and immunoprecipitated samples were run in NUPAGE 12% Bis-Tris Protein Gel at the voltage of 90V for 2 hours in MOPS buffer and then transferred to PVDF Blotting Membrane at the voltage of 100V for 100 minutes. The membrane was blocked in the buffer (5% skim milk and 0.1% Tween20 in TBS) for 1 hour at room temperature and subsequently incubated in the blocking buffer containing rabbit anti-HA (1:1000, as described above), rabbit antiLRRC37B (1:1000, as described above), mouse anti-FGF13A (1:1000, as described above), mouse anti-pan-FGF13 (1:1000; PA5-27302, ThermoFisher), mouse antibeta-actin (1:5000; MAI-140, ThermoFisher), mouse anti-pan-NAVa (1:500, as described above), rabbit anti-FLAG (1:1000; as described above), rabbit anti-SCNIB (1:500; CST 13950S, Bioke) antibodies overnight at 4°C, followed by the incubation in the blocking solution containing secondary antibody anti-Rabbit or Mouse IgG antibody conjugated with HRP at room temperature for 1 hour. Pierce ECL Western Blotting Substrate was used for signal detection.
Binding assay & affinity approximation. To estimate the FGF13A - LRRC37B affinity on the cell surface, a binding assay approach has been used as described in (Savas et al., 2015). Briefly, HEK-293T cells were plated on 10cm plates, non-transfected (6 times) or transfected with the pdisplay empty vector (6 times), LRRC37B_ECTO (6 times), LRR_ECTO (3 times) ALRR_ECTO (3 times)(described above), cultured for 24 hours, gently trypsinized and re-plated on 24 well plates and cultured for an additional 24 hours. Live cells were incubated with FGF13A ExonS-biotin at 0, 1, 5, 10, 50, 100 nM, fixed, probed with a streptavidin-HRP and reacted with TMB. The reaction was stopped with IN HCI and transferred to 96 well plates and the absorbance was measured on a plate reader at 450nm. All saturation binding calculations were performed with GraphPad Prism, One site -Specific binding, non-linear fit curve.
Pull down from rat brain extracts. Fc-Protein Purification for Mass-spectrometry was performed as described previously (Savas et al., 2014). LRRC37B (aa 28-905, containing the entire ectodomain) ectoFC protein was produced by transient transfection of HEK293T cells using PEI (Polysciences). Six hours after transfection, media was changed to OptiMEM (Invitrogen) and harvested 5 days later. Conditioned media was centrifuged, sterile filtered and run over a fast-flow Protein-G agarose (Thermo-Fisher) column. After extensive washing with wash buffer (50 mM HEPES pH 7.4, 300 mM NaCI and protease inhibitors), the column was eluted with Pierce elution buffer. Eluted fractions containing proteins were pooled and dialyzed with PBS using a Slide-A-Lyzer (Pierce) and concentrated using Amicon Ultra centrifugal units (Millipore). The integrity and purity of the purified ecto-Fc proteins was confirmed with SDS-PAGE and Coomassie staining, and concentration was determined using a Bradford protein assay. Affinity chromatography experiments were performed as previously described (Savas et al., 2014). Crude synaptosome extracts were prepared from 2-3 P21-22 rat brains per condition, homogenized in homogenization buffer (4 mM HEPES pH 7.4, 0.32 M sucrose and protease inhibitors) using a Dounce homogenizer. Homogenate was spun at 1,000 x g for 10 minutes at 4°C. Supernatant was spun at 14,000 x g for 20 minutes at 4°C. P2 crude synaptosomes were re-suspended in Extraction Buffer (50mmHEPES pH 7.4, O.lMNaCI, 2 mM CaCI2, 2.5 mM MgCI2 and protease inhibitors), extracted with 1% Triton X-100 for 2 hours and centrifuged at 100,000 x g for 1 hour at 4 C to pellet insoluble material. Fast-flow Protein- A Sepharose beads (GE Healthcare) (250 pl slurry) pre-bound in Extraction Buffer to 100 pg human Fc or LRRC37-Fc were added to the supernatant and rotated overnight at 4°C. Beads were packed into Polyprep chromatography columns (BioRad) and washed with 50 mL of high-salt wash buffer (50 mM HEPES pH 7.4, 300 mM NaCI, 0.1 mM CaCI2, 5% glycerol and protease inhibitors), followed by a wash with 10 mL low-salt wash buffer (50 mM HEPES pH 7.4, 150 mM NaCI, 0.1 mM CaCI2, 5% glycerol and protease inhibitors). Bound proteins were eluted from the beads by incubation with Pierce elution buffer and TCAprecipitated overnight. The precipitate was re-suspended in 8 M Urea with ProteaseMax (Promega) per the manufacturer's instruction. The samples were subsequently reduced by 20-minute incubation with 5mM TCEPO (tris(2carboxyethyl)phosphine) at RT and alkylated in the dark by treatment with 10 mM lodoacetamide for 20 additional minutes. The proteins were digested overnight at 37°C with Sequencing Grade Modified Trypsin (Promega) and the reaction was stopped by acidification. Mass spectrometry analysis was performed by the VIB Proteomics Core (Ghent, Belgium).
Statistical analysis. Data are represented as mean + standard error of the mean (SEM), individual values with mean, or if when detailed in the Figures with median + SE median. Unpaired Mann-Whitney test or paired Wilcoxon tests have been used for assessing the significance of differences in the analyses containing two conditions and two-way ANOVA in the analyses containing more than three conditions using GraphPad. See each figure for details. For mouse image quantification data, we used at least 3 neurons per animal, 3 animals per litter (littermates comparison) and at least 3 litters. For mouse IUE electrophysiological data we used 9-15 animals per group from four litters. Peptide application was done on 2-7 animals per condition. For co-immunoprecipitations, experiments with HEK-293T cells have been performed >3 times, experiments from mouse in vivo samples > 2 times and human in vivo samples >3 times. For human AIS intensity profile data we analysed 60 neurons per patient, 3 patients (7-38 years old); and for electrophysiology, 4-6 neurons per patient and 7 patients (4- 62 years old).

Claims

1. A modulator of the interaction between a LRRC37B protein and a NAVI.6 voltage-gated sodium channel for use as a medicine.
2. The modulator according to claim 1 for use according to claim 1, wherein the modulator is a peptide comprising a FGF13A fragment or a peptidomimetic thereof, preferably a FGF13A S-fragment or a peptidomimetic thereof.
3. The modulator according to any one of claims 1 or 2 for use according to claim 1, wherein said modulator comprises a FGF13A fragment, said FGF13A fragment comprising SEQ ID No.15 or SEQ ID No. 4 or a peptidomimetic thereof.
4. The modulator according to any one of claims 1 to 3 for use according to claim 1, wherein said modulator comprises a FGF13A fragment, said FGF13A fragment consisting of SEQ ID No.15 or SEQ ID No. 4 or a peptidomimetic thereof.
5. An isolated FGF13A protein fragment comprising or consisting of the amino acid sequence as depicted in SEQ ID No. 15 or SEQ ID No. 4.
6. An isolated LRRC37B protein fragment comprising or consisting of the amino acid sequence as depicted in SEQ ID No. 8 or SEQ ID No. 12.
7. A peptidomimetic generated from the protein fragment according to any one of claims 5 or 6.
8. A nucleic acid molecule encoding the isolated protein fragment of any one of claims 5 or 6.
9. A host cell recombinantly expressing the nucleic acid molecule according to claim 8.
10. The isolated FGF13A protein fragment according to claim 5, the isolated LRRC37B protein fragment according to claim 6, the peptidomimetic according to claim 7 or the nucleic acid molecule according to claim 8 for use as a medicine.
11. The modulator according to any one of claims 1 to 4 or the isolated FGF13A protein fragment according to claim 5, the isolated LRRC37B protein fragment according to claim 6, the peptidomimetic according to claim 7 or the nucleic acid molecule according to claim 8 for use to treat neuron excitability disorders.
12. An in vitro method of identifying a modulator of neuronal excitability, said method comprising the steps of:
- providing a LRRC37B protein, or a fragment thereof comprising a LRR domain as comprising or consisting of SEQ ID No. 8;
- providing a FGF13A protein, or a fragment thereof comprising or consisting of SEQ ID No. 15 or SEQ ID No. 4;
- contacting the LRRC37B protein or the fragment thereof with the FGF13A protein or the fragment thereof in the presence or absence of a test compound; and - identifying as said modulator of neuronal excitability the test compound that is, compared to identical conditions in absence of the test compound, statistically significantly reducing or increasing the binding of the FGF13A protein or fragment thereof to the LRRC37B protein or fragment thereof.
13. An in vitro method of identifying an LRRC37B binding peptide, said method comprises the steps of: i) Providing a LRRC37B protein, or a fragment thereof comprising or consisting of a LRR domain as depicted in SEQ ID No. 8; ii) providing a FGF13A protein fragment derived from the FGF13A S-fragment as depicted in SEQ ID No. 4, preferably said FGF13A protein fragment comprising or consisting of SEQ ID No. 15 or generating a peptidomimetic derived from a FGF13A S-fragment as depicted in SEQ ID No. 4, preferably said peptidomimetic comprising SEQ ID No. 15; iii) contacting the LRRC37B protein or the fragment thereof provided in i) with the FGF13A protein fragment or peptidomimetic provided in ii); iv) identifying as the LRRC37B binding peptide from iii) FGF13A protein fragment or peptidomimetic thereof that shows a statistically significantly increased binding to the LRRC37B protein or the fragment thereof provided in step i) compared to the binding between the FGF13A S-fragment as depicted in SEQ ID No. 4 and the LRRC37B protein or the fragment thereof. 4. The method according to any one of claims 12 to 13, wherein the binding of the LRRC37B protein or the fragment thereof to the FGF13A protein or the fragment thereof is determined by immunologic or radiologic detection, co-sedimentation, co-immunoprecipitation or electron microscopy. 5. The method according to any one of claims 12 to 14, wherein the LRRC37B protein or the fragment thereof is provided by cells expressing the LRRC37B protein or the fragment thereof. 6. The method according to claim 15, wherein the FGF13A protein or the fragment or peptidomimetic thereof is administered extracellularly to the cells expressing the LRRC37B protein or the fragment thereof. 7. A method of identifying a modulator of neuron excitability comprising the steps of: generating a peptide consisting of less than 30 contiguous amino acids wherein the peptide is a fragment of FGF13A as depicted in SEQ ID No. 4, preferably as depicted in SEQ ID No. 15, or generating a peptidomimetic of said peptide; providing a NAVI.6 protein as depicted in SEQ ID No. 14 or a functional fragment thereof; contacting a NAVI.6 protein or the fragment thereof with said peptide or peptidomimetic; identifying as a modulator of neuron excitability a peptide or peptidomimetic that, compared to identical conditions but in absence of the peptide or peptidomimetic, statistically significantly reduces or increases the activity of the NAVI.6 protein. The method according to claim 17, wherein the NAVI.6 or the functional fragment thereof is provided by cells expressing NAVI.6 or the functional fragment thereof The method according to claim 18, wherein the cells are electrically excitable cells. A modulator of the interaction between LRRC37B and SCN1B for use as a medicine. An in vitro method of identifying a modulator of neuronal excitability, said method comprising the steps of:
- providing a LRRC37B protein, or a fragment thereof comprising a LRRC37B specific domain as depicted in SEQ ID No. 12;
- providing a SCN1B protein as depicted in SEQ ID No. 10 or a fragment thereof;
- contacting the LRRC37B protein or the fragment thereof with the SCN1B protein or the fragment thereof in the presence or absence of a test compound; and
- identifying as modulator of neuronal excitability a test compound that is, compared to identical conditions but for the absence of said compound, statistically significantly reducing or increasing the binding of the SCN1B protein or fragment thereof to LRRC37B or fragment thereof. The method according to claim 21, wherein the binding of the LRRC37B protein or the fragment thereof to the SCN1B protein or the fragment thereof is determined by immunologic or radiologic detection, co-sedimentation, co-immunoprecipitation or electron microscopy. The method according to any one of claims 21 to 22, wherein LRRC37B or the fragment thereof is provided by cells expressing LRCC37B or the fragment thereof. A method of identifying a modulator of neuron excitability is provided comprising the steps of: generating a peptide consisting of less than 30 contiguous amino acids wherein the peptide is a fragment of a LRRC37 B-specific domain as depicted in SEQ ID No. 12 or generating a peptidomimetic of said peptide; providing a SCN1B protein as depicted in SEQ ID No. 10 or a functional fragment thereof; contacting the SCN1B protein with the peptide or peptidomimetic or with the LRRC37 B- specific domain as depicted in SEQ ID No. 12; identifying as a modulator of neuron excitability a peptide or peptidomimetic that, compared to identical conditions shows a statistically significantly increased binding to SCN1B compared to the LRRC37 B-specific domain as depicted in SEQ ID No. 12.
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