WO2020260670A1 - Composés destinés à être utilisés dans le traitement du syndrome de dravet et d'autres troubles - Google Patents

Composés destinés à être utilisés dans le traitement du syndrome de dravet et d'autres troubles Download PDF

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WO2020260670A1
WO2020260670A1 PCT/EP2020/068166 EP2020068166W WO2020260670A1 WO 2020260670 A1 WO2020260670 A1 WO 2020260670A1 EP 2020068166 W EP2020068166 W EP 2020068166W WO 2020260670 A1 WO2020260670 A1 WO 2020260670A1
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Prior art keywords
substitution
jztx
peptide
seq
peptidomimetic
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PCT/EP2020/068166
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English (en)
Inventor
Rémy BEROUD
Charles Cohen
Sophia Lin
Kuldip KHAKH
Michel De Waard
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Smartox
Xenon Pharmaceuticals Inc.
Inserm (Institut National De La Sante Et De La Recherche Medicale)
Universite De Nantes
Centre National De La Recherche Scientifique (Cnrs)
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Publication of WO2020260670A1 publication Critical patent/WO2020260670A1/fr

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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
    • A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • 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
    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders

Definitions

  • the present invention relates to compounds, which are useful for enhancing Na v l.l activity, and more particularly for use in the treatment or prevention of a disorder susceptible of being improved by activating the Na v l.l channel, such as Dravet syndrome.
  • Ion channels are pore-forming membrane proteins present in the membranes of excitable cells, which allow ions to pass through the channel pore. They can be classified by type of ions, by gating (i.e. the nature of the trigger that opens and closes the channel) and by cellular and tissue distribution. Voltage-gated sodium channels (Na v ) open in response to a membrane potential, in order to allow the entry of sodium ions (Na + ). They are largely responsible for action potential initiation and propagation, in most neurons. Voltage-gated sodium channels have three main conformational states: closed (or resting), open (or activated or active) and inactivated (or inactive). Only open/active state is ion permeable.
  • the cell membrane is polarized, the pore is closed, while the intracellular gating mechanism remains capable to open; the Na + ions cannot flow through the channel.
  • the membrane depolarizes, opening the pore of the channel and allowing Na + to enter the cell.
  • a refractory period occurs, while the membrane is still depolarized; the gating mechanism closes the intracellular portion of the pore, rendering the channel inactive (i.e. the channel does not open in response to a new signal).
  • activation/deactivation between open and closed, respectively
  • inactivation/reactivation between inactivated and open, respectively
  • closed-state inactivation/recovery from inactivation between inactivated and closed, respectively.
  • fast and slow there are at least two distinct kinetic classes of inactivation termed fast and slow.
  • Epilepsy in its wide spectrum (as reviewed in Catterall et al. (2010), J Physiol 588: 1849-1859), including febrile seizures, generalised epilepsy with febrile seizures plus (GEFS+), Dravet syndrome (previously known as Severe Myoclonic Epilepsy of Infancy (SMEI)), borderline severe myoclonic epilepsy of infancy (borderline SMEI), familial febrile seizures 3A (FEB3A), intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC) and temporal epilepsy ; - Alzheimer's disease and its associated cognitive dysfunction (Martinez-Losa et al. (2016), Neuron 98, 75-89; Verret et al. (2012), Cell 149: 708-721) ;
  • Autism spectrum disorders including autism itself (and in particular, familial autism) (D'Gama et al. (2015), Neuron 88 : 910-917) ;
  • Schizophrenia von Schoubye et al. (2016), Neuroscience Letters 662 : 29-35); Familial hemiplegic migraines (FHM); and
  • IBD Inflammatory Bowel Disease
  • Dravet syndrome is a rare and disastrous form of epilepsy that begins in the first year of life.
  • the frequency of occurrence is 1 out of 15,700 individuals in the United States, placing this disease in the "rare" category.
  • 80 to 90% of the patients have a mutation in one allele of the SCN1A gene, which can be missense (40%), nonsense (20%), frameshift (20%), duplications/deletions (7%), or splice site mutations (10%) (for a complete list of mutations, see https://ghr.nlm.nih.gov/primer/mutationsanddisorders/possiblemutations).
  • Dravet syndrome patients comprise prolonged and frequent seizures, developmental delays, trouble of movement and balance, delayed language performance, sleeping problems, chronic infections, and abnormalities in sensory integration and of the autonomic nervous system.
  • SUADP Sudden Unexpected Death in Epilepsy
  • First line treatment involves valproic acid, and if not effective, then clobazam.
  • Second line treatment comprises stiripentol in addition to valproic acid and clobazam, or topiramate.
  • the third line of treatment comprises the addition of an anti-epileptic drug such as clonazepam, phenobarbital, levetiracetam, zonisamide, or ethosuximide. While this polypharmacy with anticonvulsant medications is the standard-of-care, the outcome remains poor, thus reflecting the urgent need for new therapeutic options.
  • Several compounds are currently in clinical trials (e.g.
  • all anti-epileptic drugs active as sodium channel blockers carbamazepine, oxcarbazepine, lamotrigine, vigabatrin, rufinamide, phenytoin, fosphenytoin
  • the different isoforms of voltage-dependent sodium channels from eukaryotic cells differ in terms of biophysical properties, tissue distribution and cellular function.
  • the Na v l.l and Na v 1.2 isoforms are expressed in neurons, while Na v 1.5 is predominantly expressed in cardiac myocytes.
  • all sodium channels of the Na v l family consist of a highly processed a subunit that is approximately 260-280 kDa (and which consists of up to 2600 amino acids), and is associated with one or more accessory subunits (b ⁇ , b2, b3 and b4) in certain tissues.
  • the a subunit consists of four homologous domains (l-IV), each comprising six transmembrane segments (S1-S6), for a total of 24 transmembrane segments. Between S5 and S6, there is a hairpin-like P-loop that comprises part of the channel pore.
  • the accessory subunits b ⁇ , b2 and bB each span the membrane once.
  • the accessory b2 subunit is covalently linked to the a subunit by disulfide bonds, and the b ⁇ and bB subunits are non-covalently attached and expressed in a complementary fashion, so that a subunits are associated with either b ⁇ or b3.
  • Hmla is a 35-residue peptide having a sequence of SEQ ID NO : 7, and containing three disulfide bridges (between cysteines at position 2 and 16, between cysteines at positions 9 and 21, and between cysteines at positions 15 and 28, respectively), which has been identified from the venom of the spider Heteroscodra maculata, and is disclosed in the patent application WO2017096431.
  • Hmla has been described as an activator of Na v l.l, which acts by slowing both the fast and slow inactivation properties of Na v l.l, by a two-point interaction with the voltage-sensor of Na v l.l domain IV (Osteen et al (2016), Nature 534:494-499).
  • Hmla increases the Na + peak current and is able to produce persistent current at depolarized potentials.
  • This peptide was shown to rescue Dravet syndrome mice from seizures and premature death (Richards et al (2016), Proc Natl Acad Sci 115:E8077-E8085).
  • the main drawback of Hmla is that it also activates the Na v 1.2 isoform, also involved in epilepsy, which means that several unwanted side effects are to be expected with this peptide.
  • Jingzhaotoxin 34 (JzTx-34) has been previously identified from the venom of the tarantula Chilobrachys guangxiensis, also known as Chilobrachys jingzhao (Chen et al. (2009), Peptides 30: 1042-1048).
  • JzTx-34 is a 35-residue peptide of SEQ ID NO: 1, having a molecular weight of 4147.8 Da, which contains three disulfide bridges, between cysteines at position 2 and 16, between cysteines at positions 9 and 21, and between cysteines at positions 15 and 29, respectively.
  • JzTx-34 was shown to inhibit tetrodotoxin-sensitive sodium channels while having no significant effects on tetrodotoxin-resistant sodium channels on rat dorsal root ganglion neurons, in a whole-cell patch-clamp assay. It was later reported to act selectively on Na v 1.7 (and to a lesser extent on Na v 1.3), with little effect on other sodium channel subtypes, such as Na v l.l (Zeng et al (2016), Toxins 10, E64).
  • JzTx-34 and its peptidomimetics are actually potent and selective activators of Na v l.l, as confirmed by two independent laboratories, and are therefore particularly useful for the treatment or the prevention of a disorder susceptible of being improved by activating Na v l.l channel.
  • they may correct the haplo-insufficiency triggered by de novo mutations of one allele of SCN1A in Dravet syndrome patients. They act by both activating Na v l.l and slowing down its inactivation.
  • the present invention relates to a compound, selected in the group consisting of :
  • ACREWLGGCSKDADCCAHLECRKKWPYHCVWDWTV (SEQ ID NO: 1), and containing three disulfide bridges, between cysteines at position 2 and 16, between cysteines at positions 9 and 21, and between cysteines at positions 15 and 29, respectively;
  • a peptidomimetic thereof for use in the treatment or prevention of a disorder susceptible of being improved by activating Na v l.l channel.
  • a peptide refers to a polymer of amino acid residues, which are linked together by amide bonds.
  • Amino acids are generally L-amino acids, unless stated otherwise. Amino acids can be naturally occurring or non-naturally occurring.
  • a peptide generally consists of fewer than 200 amino acids, preferably fewer than 100 amino acids, more preferably fewer than 50 amino acids.
  • the term "peptide" also includes the possibility of typical post-translational modifications, such as disulfides bonds between cysteine residues ;
  • a peptidomimetic of the peptide JzTx-34 is defined as a molecule or compound, excluding the peptide JzTx-34 itself, comprising the minimal active part derived from the pharmacophore of JzTx-34 (e.g. lateral chains or portions of lateral chains of a few key amino acids of the pharmacophore of JzTx-34), arranged in the same 3D configuration, and which are able to bind onto the same binding site of Na v l.l as JzTx-34 and induce Na v l.l channel activation.
  • the minimal active part derived from the pharmacophore of JzTx-34 e.g. lateral chains or portions of lateral chains of a few key amino acids of the pharmacophore of JzTx-34
  • the pharmacophore of JzTx-34 is defined as the set of amino acids, arranged in a particular 3D configuration, which are able to induce Na v l.l channel activation.
  • a peptidomimetic of the peptide JzTx-34 would be expected to displace JzTx-34 binding from its site on Na v l.l.
  • Peptidomimetics of the peptide JzTx-34 can be of any nature, such as peptides, modified peptides, pseudopeptides, proteins or small organic molecules. Examples of possible peptidomimetics can be found for instance in Pelay-Gimeno et al (2015), Angew. Chem. Int. Ed. 54, 8896-8927 ;
  • the percentage of sequence identity between a given sequence (test sequence) and another sequence (reference sequence) is determined by methods well- known to a skilled person.
  • the two sequences are first optimally aligned.
  • the two sequences can be of the same size, or of different sizes. In some cases, it may be necessary to add "gaps" in one of the sequence, in order to allow an optimal alignment with the other sequence.
  • 5 sequences can for instance be carried out using the Smith and Waterman algorithm (Smith and Waterman (1981), J. Theor. Biol., 91 (2): 370-380), the Needleman and Wunsch algorithm (Needleman and Wunsch (1972), J. Mol. Biol, 48(3) : 443-453) or the Pearson and Lipman method (Pearson and Lipman (1988), Proc. Natl. Acad. Sri. U. S.A., 85(5) : 2444-2448).
  • a conservative substitution corresponds to the replacement of an amino acid by
  • 25 another amino acid having similar physicochemical properties (such as size, charge and/or hydrophobicity).
  • This similarity can be assessed in terms of physicochemical distance between two amino acids, using for instance the Grantham's distance (Grantham (1974), Science, 185 (4154): 862-864), the Sneath's index (Sneath (1966), Journal of Theoretical Biology, 12 (2) : 157-195),
  • the term "disorder” refers to an abnormality in the physical state of the body as a whole or one of its parts ;
  • activating Na v l.l channel and “enhancing Na v l.l activity” (and variants thereof) refer to an increase in the entry of Na + ions through Na v l.l, which can, for instance, occur due to enhancement of the transition of Na v l.l from a resting to an activated state (e.g. through the use of molecules that activate Na v l.l) and/or due to the delay, slowing, blocking or prevention of inactivation of Na v l.l (e.g. through the use of molecules that slow-down the inactivation of Na v l.l and/or promote longer openings), and/or due to the alteration of voltage-dependence of activation or inactivation ;
  • a “disorder susceptible of being improved by activating Na v l.l channel” is a disorder characterized by a functional deficit of Na v l.l mediated sodium entry into cells, as can be assessed by methods well-known to the skilled person, such as patch-clamp techniques or the use of intracellular fluorescent dyes sensitive to the concentration of Na + .
  • the present invention thus relates to a compound, selected in the group consisting of the peptide JzTx-34 and a peptidomimetic thereof, for use in the treatment or prevention of a disorder susceptible of being improved by activating Navl.l channel.
  • the compound for use as described above is the peptide JzTx-34.
  • the compound for use as described above is a peptidomimetic of the peptide JzTx-34.
  • This peptidomimetic may possess the following characteristics, taken individually or in combination, as can be determined by a skilled person:
  • the peptidomimetic of the peptide JzTx-34 comprises or consists essentially of the peptide JzTx-34 ;
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34, by substitution of one or more amino acids in SEQ ID NO: 1, wherein the resulting amino acid sequence has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 1.
  • the peptidomimetic of the peptide JzTx-34 has at least 70% sequence identity with SEQ ID NO: 1, still preferably at least 80% sequence identity with SEQ ID NO: 1. More preferably, the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34 by conservative substitution of one or more amino acid(s) of SEQ ID NO: 1. Even more preferably, the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34 by substitution of one or more amino acids of SEQ ID NO: 1, selected from the group consisting of:
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34, by insertion and/or deletion of one or more amino acids in SEQ ID NO: 1, wherein the amino acids to be deleted do not include cysteines.
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34 by the deletion of a maximum of one amino acid at the N-terminus, and/or the deletion of a maximum of six, five, four, three, two, one amino acid(s) at the C- terminus, compared to SEQ ID NO: 1.
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34 by the deletion of a maximum of two amino acids, more preferably a maximum of one amino acid, and/or the insertion of a maximum of two amino acids, more preferably a maximum of one amino acid, compared to SEQ ID NO: 1 ;
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34 by substitution of one or more L-amino acid(s) by corresponding D-amino acid(s).
  • the amino acid sequence of the peptide JzTx- 34 is rearranged in the reverse order (retro-inverso peptides) ;
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34 by replacement of one or more disulfide bond(s) by diselenide, lanthionine, lactam or dimethylene bond(s).
  • a disulfide bond is replaced by a diselenide bond ;
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34 by modifications at the N-terminus and/or at the C-terminus.
  • modifications at the N-terminus can be selected from the group consisting of acetylation, alkyne functionalization, coupling with a spacer (typically, 6- IB aminohexanoic acid, PEG or 3,6-dioxaoctanoic acid), a carrier, a fluorescent dye, an affinity tag, pyroglutamic acid, a fatty acid.
  • modifications at the C-terminus can be selected from the group consisting of amidation, coupling with a spacer (typically, 6-aminohexanoic acid, PEG or 3,6-dioxaoctanoic acid), addition of an extra lysine that can be labelled in side chain (e.g. with a fluorescent dye, an affinity tag, a fatty acid) ;
  • a spacer typically, 6-aminohexanoic acid, PEG or 3,6-dioxaoctanoic acid
  • an extra lysine that can be labelled in side chain e.g. with a fluorescent dye, an affinity tag, a fatty acid
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34 by alkylation of one or more amide bond(s) ;
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34 by connection of the side chain of one or more amino acid(s) to the nitrogen of the peptide backbone, instead of the a-carbon (peptoids) ;
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34 by replacement of a-carbon of one or more amino acid(s) by nitrogen (azapeptides and azatides).
  • JzTx-34 The most critical residues in JzTx-34, involved in the binding and activation of Na v l.l, are W5, L6, R22, P26 and W33 ; other important residues are H18, K23, W25, H28, D32 and T34.
  • Other residues of JzTx-34, which are important for the activation on Na v l.l, although not involved in the binding to Na v l.l, are L19, V30 and W31.
  • one or more of these residues is(are) preferably replaced by another amino acid residue, as follows :
  • L6 lie, Val, Ala, Gly, Phe (more preferably lie or Val)
  • L19 lie, Val, Ala, Gly, Phe (more preferably lie or Val)
  • R22 Lys, His, Gin (more preferably Lys)
  • K23 Arg, His, Asn (more preferably Arg)
  • W25 Tyr, Phe, Val, Thr, Ser, Met, Leu, lie, Gin, Asn (more preferably Tyr or Phe) - P26 : Val, Phe, Leu, lie, Gly
  • V30 lie, Leu, Ala, Gly, Phe (more preferably lie or Leu)
  • W31 Tyr, Phe, Val, Thr, Ser, Met, Leu, lie, Gin, Asn (more preferably Tyr or Phe)
  • D32 Glu, Asn, Gin (more preferably Glu)
  • W33 Tyr, Phe, Val, Thr, Ser, Met, Leu, lie, Gin, Asn (more preferably Tyr or Phe)
  • T34 Ser, Cys (more preferably Ser)
  • Remaining amino acids at other positions in SEQ ID NO: 1 are less important for activation of Na v l.l, and can be replaced by any other amino acid. Although it can also be replaced by Asp, it is particularly advantageous to replace E4 by another amino acid residue which is not negatively charged (among natural amino acids, this means all other amino acids, except Asp).
  • the present invention relates to a peptidomimetic of the peptide JzTx- 34, for use as defined above, comprising, consisting of, or consisting essentially of any of the following amino acid sequences :
  • ACRELLGGCSKDSDCCAHLECRKKWPYHCVWDWTI (SEQ ID NO: 2) ACRELLGGCSKDSDCCAHLECRKKWPYHCVWDWTF (SEQ ID NO: 3) CRYLMGGCSKDGDCCEHLVCRTKWPYHCVWDWTF (SEQ ID NO: 4) CTKLLGGCKTDAECCPHLGCRKKWPYHCGWD (SEQ ID NO: 5), or CTKLLGGCTKDSECCPHLGCRKKWPYHCGWDGT (SEQ ID NO: 6)
  • these peptides may advantageously be amidated at the C-terminus.
  • the peptide JzTx-34 and its peptidomimetics can be obtained by different means, which are well known to a skilled person.
  • peptides can be obtained by chemical synthesis ("synthetic peptide") or by recombinant methods (“recombinant peptide”), and some of them can also be isolated from natural sources (“isolated peptide").
  • the peptide JzTx-34 can for instance be purified from the crude venom of the tarantula Chilobrachys guangxiensis, by a multiple step fractionation procedure, involving RP- HPLC and cation exchange, using a pharmacological guided bioassay on various isoforms of sodium channels (Na v l.l, Na v 1.2, Na v 1.5, Na v 1.6 and Na v 1.7).
  • the peptides of the present invention may also be prepared by chemical synthesis, using standard peptide synthesis methods, such as solution synthesis or solid phase synthesis, followed by oxidative disulfide bond formation.
  • the chemical synthesis of the peptides of the invention may be performed manually or using an automated synthesiser.
  • the linear peptides may be synthesised using solid phase peptide synthesis using either Boc or Fmoc chemistry, as described in Merrifield (1963) J Am Chem Soc, 85(14): 2149-2154; Schnolzer, et al. (1992) Int J Pept Protein Res, 40: 180-193 and Cardosa, et al. (2015) Mol Pharmacol, 88(2): 291-303.
  • the linear peptides are purified using suitable methods, such as preparative chromatography.
  • the purified linear peptides are then oxidized in buffered systems to form the disulfide bonds or using directed fold with various Cys protecting groups, followed by purification using a suitable means, such as preparative chromatography.
  • a synthetic method involving selective disulfide bond formation may be used as described in, for example, Kent, et al. (1998) Biopolymers, 46: 53-63.
  • the peptide may be cyclised.
  • Disulfide bond replacement with diselenide, lanthionine, lactam or dimethylene bonds may be prepared using methods known in the art, for example, as described in Muttenthaler and Alewood (2008) J Pept Sci, 14(12): 1223-1239; Li, et al. (2002) Current Organic Chemistry, 6: 411-440; and Fazio, et al. (2005) Biopolymers (Peptide Science), 84(2):
  • the peptides of the present invention can also be prepared using recombinant DNA techniques. For example, they may be prepared by a procedure including the steps of:
  • a construct typically, an expression vector
  • a construct comprising a polynucleotide sequence that encodes the peptide of the invention and that is operably linked to a regulatory element, such as transcriptional and translational regulatory nucleic acids ;
  • the polynucleotide sequence that encodes said peptide can for instance be genomic DNA or cDNA obtained by retro-transcription of mRNA, wherein nucleic acids (genomic DNA, mRNA) encoding prepropeptide precursors of the mature peptides can be isolated from venomous glands of these animals (Chen et al (2008), Cell. Mol.
  • the peptide JzTx-34 and peptidomimetics thereof may thus be used in the treatment or prevention of a disorder susceptible of being improved by activating Na v l.l channel.
  • the invention relates to a peptide JzTx-34 and peptidomimetics thereof, for use in the treatment or prevention of a disorder susceptible of being improved by activating Na v l.l channel, wherein the disorder susceptible of being improved by activating Na v l.l channel is associated with or triggered by loss-of-function mutations in the SCN1A gene.
  • Said mutations can be monoallelic or biallelic.
  • the disorder susceptible of being improved by activating Na v l.l channel is selected from the group consisting of epilepsy (and in particular febrile seizures, generalized epilepsy with febrile seizures plus, Dravet syndrome, borderline severe myoclonic epilepsy of infancy, familial febrile seizures 3A, intractable childhood epilepsy with generalized tonic clonic seizures and temporal epilepsy), Alzheimer's disease and its associated cognitive dysfunction, autism spectrum disorders (and in particular, autism, and more particularly, familial autism), schizophrenia, familial hemiplegic migraines and Inflammatory Bowel Disease (IBD).
  • the disorder susceptible of being improved by activating Na v l.l channel is Dravet syndrome.
  • the individual having a disorder susceptible of being improved by activating Na v l.l channel can be a human or an animal, such as a cat, a dog, a horse, a rabbit, a hamster, a pig, a bovine (e.g. a cow), or a caprine (e.g. a goat).
  • said individual is a human.
  • the present invention also relates to a pharmaceutical composition comprising a peptide JzTx-34 or a peptidomimetic thereof, as defined above, for use in the treatment or prevention of a disorder susceptible of being improved by activating Na v l.l channel.
  • the pharmaceutical composition according to the present invention may also contain pharmaceutically acceptable carriers, vehicles and/or diluents including any and all solvents, dispersion media, coatings, antimicrobial agents, isotonic and absorption delaying agents, as can be determined by a skilled person.
  • the pharmaceutical compositions of the present invention may be administered through a variety of routes, including, but not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, intracerebral, intracerebroventricular, intrathecal, epidural, oral, ocular, sublingual, intranasal and inhalation administration; especially intracerebral, intracerebroventricular, intrathecal and epidural administration.
  • the present invention further relates to the use of a peptide JzTx-34 or a peptidomimetic thereof, as defined above, for preparing a medicinal product intended for the treatment or prevention of a disorder susceptible of being improved by activating Na v l.l channel, more particularly a disorder selected among the disorders disclosed above, and even more particularly Dravet syndrome.
  • the present invention further relates to a method of treating or preventing a disorder susceptible of being improved by activating Na v l.l channel, more particularly a disorder selected among the disorders disclosed above, and even more particularly Dravet syndrome, in a subject suffering or susceptible to suffer from said disorder, by administering a peptide JzTx-34 or a peptidomimetic thereof, as defined above.
  • the present invention also relates to a peptidomimetic of the peptide JzTx- 34, which is derived from said peptide JzTx-34, by one or more of the following modifications:
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34, by substitution, insertion and/or deletion of one or more amino acids in SEQ ID NO: 1, wherein the amino acids to be deleted do not include cysteines, and wherein the resulting amino acid sequence has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 1, and does not comprise, consist of or consist essentially of the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 ;
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34 by substitution of one or more L-amino acid(s) by corresponding D-amino acid(s) ;
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34 by replacement of one or more disulfide bond(s) by diselenide, lanthionine, lactam or dimethylene bond(s) ;
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34 by modifications at the N-terminus ;
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34 by alkylation of one or more amide bond(s) ;
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34 by connection of the side chain of one or more amino acid(s) to the nitrogen of the peptide backbone, instead of the a-carbon ;
  • the peptidomimetic of the peptide JzTx-34 is derived from the peptide JzTx-34 by replacement of a-carbon of one or more amino acid(s) by nitrogen.
  • the peptidomimetic of the peptide JzTx-34 is derived from said peptide JzTx-34 by substitution, more preferably conservative substitution, of one or more amino acid(s) of SEQ ID NO: 1, wherein the resulting amino acid sequence has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 1, and does not comprise, consist of or consist essentially of the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • the peptidomimetic of the peptide JzTx-34 as described above is derived from said peptide JzTx-34 by substitution of one or more amino acid(s) of SEQ ID NO: 1, selected from the group consisting of:
  • amino acid residues in SEQ ID NO: 1 is(are) preferably replaced by another amino acid residue, as follows :
  • W5 Leu, lie, Phe, Tyr, Val, Thr, Ser, Met, Asn, Gin (more preferably Leu, Tyr or Phe)
  • L6 lie, Val, Ala, Gly, Phe (more preferably lie or Val)
  • L19 lie, Val, Ala, Gly, Phe (more preferably lie or Val)
  • R22 Lys, His, Gin (more preferably Lys)
  • K23 Arg, His, Asn (more preferably Arg)
  • W25 Tyr, Phe, Val, Thr, Ser, Met, Leu, lie, Gin, Asn (more preferably Tyr or Phe) - P26 : Val, Phe, Leu, lie, Gly
  • V30 lie, Leu, Ala, Gly, Phe (more preferably lie or Leu)
  • W31 Tyr, Phe, Val, Thr, Ser, Met, Leu, lie, Gin, Asn (more preferably Tyr or Phe)
  • D32 Glu, Asn, Gin (more preferably Glu)
  • W33 Tyr, Phe, Val, Thr, Ser, Met, Leu, lie, Gin, Asn (more preferably Tyr or Phe)
  • T34 Ser, Cys (more preferably Ser)
  • Remaining amino acids at other positions in SEQ ID NO: 1 are less important for activation of Na v l.l, and can be replaced by any other amino acid. Although it can also be replaced by Asp, it is particularly advantageous to replace E4 by another amino acid residue which is not negatively charged (among natural amino acids, this means all other amino acids, except Asp).
  • the present invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a peptidomimetic of the peptide JzTx-34 as described above.
  • the present invention further relates to a peptidomimetic of the peptide JzTx-34 as described above, for use as a medicament.
  • the present invention further relates to a process of producing a peptidomimetic of the peptide JzTx-34 as described above, by chemical synthesis or by recombinant methods.
  • the present invention further relates to:
  • an expression cassette comprising said nucleic acid molecule
  • a vector comprising said nucleic acid molecule or expression cassette ;
  • said vector is a plasmid
  • Figure 1 HPLC chromatogram using a C18 analytical column and a UV detection at 214 nm, illustrating the similar retention times of the isolated JzTx-34 peptide (purified natural) and the synthetic JzTx-34 peptide. Peak of elution is between 22.6 and
  • FIG. 2 Effect of JzTx-34 on Na + influx on various human Na v (hNa v ) isoforms: Na + influx inhibition (upward) or activation (downward).
  • the JzTx-34 peptide activated hNa v l.l with an EC50 value of 75 nM, while it inhibited various channel isoforms with a lower affinity (169 nM for hNavl.2, 1317 nM for hNavl.5, 3970 nM for hNavl.7 and 7022 nM for hNavl.6).
  • FIG. 3 (top panel ) The trace currents recorded by patch clamp illustrate the mechanism of action of JzTx-34 on hNa v l.l.
  • the JzTx-34 peptide delayed the channel inactivation as seen by a persistent current remaining at the end of a 10-ms depolarizing pulse to 0 mV. In the absence of the peptide, Na v l.l was completely inactivated.
  • ( lower panel ) Time course of development of this delayed inactivation by JzTx-34 peptide (at 74 nM) was measured by the integral of the current during the test depolarization. As shown, at this concentration, JzTx-34 stimulated only hNavl.l and had no effect on the other isoforms tested.
  • Figure 4 Effect of the JzTx-34 concentration on current activation, based on the measurement of current integrals as in Figure 3. The same EC50 value of 75 nM was found for current activation by reduction of channel inactivation. In comparison, none of the other isoforms enhanced Na + influx in cells.
  • Figure 5 Representative current traces illustrating the effect of JzTx-34 at different concentrations on the inactivation of hNavl.l and the induction of a persistent current, compared to control. As can be seen in this figure, a persistent current could be induced at a concentration as low as 10 nM.
  • Figures 7-1 to 7-8 (together.
  • Figure 8 Histogram of EC50 values for each JzTx-34 analogue from Ala scan, for the amidated JzTx-34 and phlotoxin la and lb analogues.
  • Y-axis is loglO representation.
  • Various shades illustrate the extent of degradation in average EC50 values along the Ala scan procedure.
  • Figure 9 3D structure of JzTx-34 showing the various amino acids, which are most important for binding and activation of Na v l.l (Trp5, Leu6, Hisl8, Arg22, Lys23, Trp25, Pro 26, His28, Asp32, Trp33, Thr34).
  • Glu20, Tyr27 and Lys24 play more minor roles. All these residues are located on the same face of the toxin, hence forming a comprehensive set of residues important for interaction with Na v l.l.
  • Figure 10 3D structure of JzTx-34 showing the position of the disulfide bridges and the fact that they structure JzTx-34.
  • Leul9, Val30 and Trp31 are burried inside the toxin and cannot therefore contribute to the binding to Na v l.l.
  • mutations of these residues are disruptive to the point to affect the global structure of the peptide.
  • FIG. 11 JzTx-34 at a concentration of 100 nM produces a potent inhibition of a chimera potassium channel (K v 2.1) in which the S3-S4 segment was replaced by the S3- S4 segment of the fourth domain of Na v l.l.
  • K v 2.1 chimera potassium channel
  • This effect indicates that JzTx34 binds onto the S3-S4 segment of domain IV of human Na v l.l to produce channel activation.
  • the chimera we see inhibition rather than activation because the coupling to gating is different in potassium channels.
  • Example 1 Purification of the peptide JzTx-34, from the venom of C. guangxiensis (isolated JzTx-34 peptide)
  • the peptide JzTx-34 consisting of SEQ ID NO: 1 and having three disulfide bridges as described previously, was purified from the crude venom of the tarantula C. guangxiensis, by a multiple step fractionation procedure, using RP-HPLC followed by cation exchange, using a pharmacological guided bioassay on various isoforms of sodium channels (Na v l.l, Na v 1.2, Na v 1.5, Na v 1.6 and Navi.7) and with the help of mass spectrometry identification.
  • Example 2 Solid-phase synthesis of the peptide JzTx-34 (synthetic JzTx-34 peptide) and of the peptide Hmla
  • Linear JzTx-34 was assembled stepwise using Fmoc solid-phase chemistry on a Symphony Synthesiser (Protein Technologies Inc.), at a 0.1 mmol scale on 2-chlorotrityl chloride resin (substitution approx. 1.6 mmol/g).
  • Fmoc protecting group was removed using 20% piperidine in DMF and free amine was coupled using tenfold excess of Fmoc amino acids and HCTU/DIEA activation in NMP/DMF (3x15 min).
  • Linear peptide was deprotected and cleaved from the resin with TFA/H 2 0/l,3-dimethoxybenzene/TIS 92.5/2.5/2.5/2.5 (vol.), then precipitated out in cold diethyl ether. The resulting white solids were washed 2 times with diethyl ether, re-suspended in H 2 0/acetonitrile and freeze dried to afford crude linear peptide.
  • the crude peptide was folded by air oxidation at 25 mM in a 200 mM ammonium acetate buffer at pH 7.8, containing 5 mM GSH and 0.5 mM GSSG. After 72 h at 4°C, the pH of the reaction mixture was adjusted to 3 and purified by preparative HPLC.
  • the peptide Hmla was also synthesized by SPPS, as a comparison. Hmla was synthesized by SPPS using Fmoc strategy on an automated synthesizer Symphony X de Gyros Protein Technologies. The first amino acid Fmoc-Ser(tBu)-OH was manually loaded on a chlorotrityl chloride polystyrene resin, and the rest of the sequence was added automatically using 10 equivalents of amino acids, 10 equivalents of coupling agent and 20 equivalents of basis and by repeating the coupling three times. A capping step was performed after the last coupling, then the Fmoc protecting group was deprotected with 20% piperidine solution in dimethylformamide.
  • Cysteines in position 2 and 16 were introduced with acetamidomethyl (Acm) protecting groups on the lateral chains, cysteines in position 9 and 21 by trityl groups and cysteines in position 15 and 28 by methoxybenzyle (Mob) groups.
  • Amino acids AspBl and Gly32 were introduced under the form of dipeptide Fmoc-Asp(OtBu)-(Dmb)Gly-OH, and the amino acids SerlO and Serll introduced under the form of pseudoproline Fmoc-Ser(tBu)- Ser(Psi(Me,Me)pro)-OH.
  • the oxidative folding of Hmla was performed in three distinct steps corresponding each to the formation of a disulfide bridge.
  • the first bridge was formed by oxidation with DMSO starting with the crude synthetic product by SPPS and corresponds to the bridge between the deprotected Cys(Trt). The product of this first bridge formation occurred with a mean yield of 25 %.
  • the second disulfide bridge was formed between the Cys(Acm) by oxidation reaction with diiodine. The product was obtained with an average yield of 40 %.
  • the third disulfide bridge was formed between the Cys(Mob) first deprotected with TFMSA.
  • the oxidation reaction with DMSO produced Hmla fully folded in its native configuration with a mean yield of 13%. This last step led to the formation of a byproduct corresponding to an isomer of Hmla. The total yield of the synthesis was thus 1.3%.
  • Example 3 Coelution profile of the synthetic JzTx-34 peptide and the isolated (native) JzTx-34 peptide
  • Figure 1 depicts the HPLC-UV traces at 214 nm of the synthetic and the isolated (native) JzTx-34 peptides, showing their perfect coelution at 22.6 min, thus confirming that the synthetic JxTx-34 peptide was identical to the isolated (native) JzTx-34 peptide.
  • the synthetic JxTx-34 peptide was used.
  • the synthetic JzTx-34 peptide obtained in Example 2 was characterized on Na + influx (as measured with a Na-sensitive fluorescent dye indicator) in various HEK293 cell lines, each expressing a Na v isoform. These cell lines were obtained as described in Bankar et a I, Cell Reports 24, 3133-3145, 2018. Briefly, human embryonic kidney cells (HEK) were used that are permanently transfected with an expression vector containing the full- length cDNA coding for the human sodium channel a-subunit, grown in culture media containing 10% FBS, 1% PSG, and 0.5 mg/mL G418 at 37°C with 5% C02.
  • HEK human embryonic kidney cells
  • GenBank accession numbers for a-subunits were: hNa v l.l (NM_006920); hNavl.2 (NM_021007), hNa v l.5 (AC137587), hNa v 1.6 (NM L99037), and hNa v 1.7 (NM_002977).
  • the human Na v 1 subunit (accession number NM_199037) was coexpressed with all a-subunits.
  • human FHF2b accession number NM_033642 was co-expressed in addition to Na v 1 to increase functional surface expression.
  • the sodium influx (in vitro) assay used will now be described.
  • This assay employs the use of the cell-permeable and sodium-sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of the sodium channel modulator, veratridine.
  • These high-throughput sodium influx assays allow for rapid profiling and characterization of sodium channel inhibitors and activators.
  • T-rex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel a- subunit and with an expression vector containing full length cDNA coding for the b ⁇ - subunit.
  • Sodium channel expressing cell lines were induced with tetracycline (1 pg/mL) and plated on 384-well PDL- coated plates at a density of 25,000 - 30,000 cells/ well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37°C, 5% C02), culture media was removed and cells were loaded with 5 mM ANG2 dye for lh in Buffer 1 (155 mM NMDG, 5 mM KCI, 2 mM CaCI2, 1 mM MgCI2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4).
  • Buffer 1 155 mM NMDG, 5 mM KCI, 2 mM CaCI2, 1 mM MgCI2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4
  • the synthetic JzTx-34 peptide enhanced Na v l.l with a concentration producing 50% of increase (EC50) of 75 nM, while it inhibited Na v 1.2, Na v 1.5, Na v 1.6 and Na v 1.7 with concentration producing 50% of inhibition (IC50) values of 169 nM, 1317 nM, 7022 nM and 3970 nM, respectively.
  • the JzTx-34 peptide displayed a higher affinity toward Na v l.l than toward the other channel isoforms tested.
  • Example 5 Effect of JzTx-34 on Na l-1 current in an HEK293 cell line expressing the channel (as compared with other sodium channels), as measured by automated patch clamp
  • the cell lines and methodology used were as described in Bankar et al., 2018. Briefly, the methods for the automated patch clamp were as follows. Sodium currents were measured using the patch-clamp technique in the whole-cell configuration using the Qube384 (Sophion A/S, Copenhagen, Denmark) automated voltage-clamp platform. "Single hole” plates were used which allows maximal series resistance and capacitance compensation in each cell. Appropriate filters for minimum seal resistance and minimum current size were applied, and series resistance was compensated >80% to yield high quality sodium channel recordings on par with manual voltage-clamp techniques. Data was collected at room temperature which corresponds to 27 ⁇ 2 °C on the recording chamber unless otherwise noted. Voltage clamp experiments were performed with a reversed sodium gradient.
  • the intracellular solution comprised (in mM): 120 NaF, 10 CsCI, 0.1 CaCI2, 2 MgCI2, 10 HEPES, 10 EGTA; adjusted to pH 7.2 with CsOH.
  • the extracellular solution comprised: 1 NaCI, 139 CholineCI, 5 KCI, 2 CaC , 1 MgC , 10 HEPES; and adjusted to pH 7.4 with NaOH, so that the total extracellular sodium was about 6 mM.
  • Osmolarity in the internal and external solutions was adjusted to 300 mOsm/kg and 310 mOsm/kg, respectively, with glucose.
  • Figure 3 shows the current recording obtained by stepping the voltage from -120 mV to 0 mV, in an HEK293 cell line expressing Na v l.l, after addition of 74 nM JzTx- 34, compared to control.
  • the effect of the JzTx-34 peptide was to cause incomplete inactivation of the sodium current, thereby generating a persistent current.
  • Both records are outward currents because the sodium gradient was reversed, so that intracellular sodium was much higher than extracellular sodium. This procedure enables improved voltage control during the depolarizations.
  • AUCs Areas under the curve
  • JzTx-34 was resolved using NMR.
  • 1 mg of JzTx-34 was dissolved in 220 pL of H2O/D2O (90/10), at pH 4.5, to a final concentration of 1,1.10 3 mol. I 1 .
  • All NMR spectra were acquired at 298K on a BRUKER 700 MHz spectrometer with a 5 mm TCI cryoprobe, namely COSY, 80ms TOCSY, 160 ms NOESY, 1 H/ 15 N SoFast-HMQC, and 1 H/ 13 C HSQC, and were processed with TopSpin3.2.
  • the 3D structure of JzTx-34 ( Figure 9) displays a distorted extended region around W5 and L6, two short strands of b-sheet L19-C21 and C29-W31, and is compacted around the 3 disulfide bridges C2-C16, C9-C21, C15-C29, thus forming the well-known ICK motif.
  • the core of the molecule is composed of residues W5, C9, C21, V30 and W31, which display a solvent accessibility close to zero. On the contrary, residues K24, W25, C29 and W33 are particularly accessible.
  • the triad of tryptophans W5/W31/W33 is responsible of out-of-range chemical shifts for a series of close protons, for example L6(6), H18(b), L19(a and d) and W31 itself (b ⁇ and indole).
  • Other protons such as W25(a), Pro26(a), H28(b and d) showed atypical chemical shifts too, influenced by Y27 and W25 itself.
  • Figure 9 shows the 3D structure of JzTx-34 and the position of different amino acid residues, classified in different groups according to the reduction in affinity when they are substituted by an alanine (Ala-scan), as determined from the AUCs in Example 8 (left : most important amino acids ; middle : important amino acids ; right : less important amino acids).
  • Amino acid residues Trp5, Leu6, Hisl8, Arg22, Lys23, Trp25, Pro26, His28, Asp32, Trp33, Thr34 in JzTx-34 are mostly responsible for the binding and activation of Na v l.l. Glu20, Tyr27 and Lys24 play minor roles.

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Abstract

La présente invention concerne des composés, qui sont utiles pour améliorer l'activité de Nav1.1, et plus particulièrement destinés à être utilisés dans le traitement ou la prévention d'un trouble susceptible d'être amélioré par activation du canal Nav1.1, tel que le syndrome de Dravet.
PCT/EP2020/068166 2019-06-26 2020-06-26 Composés destinés à être utilisés dans le traitement du syndrome de dravet et d'autres troubles WO2020260670A1 (fr)

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WO2017096431A1 (fr) 2015-12-09 2017-06-15 The University Of Queensland Molécules protéiques et procédés d'utilisation
WO2018126111A1 (fr) * 2016-12-30 2018-07-05 Goldstein Steven Alan Modulateurs hv1 et utilisations
WO2018209127A1 (fr) * 2017-05-11 2018-11-15 Los Angeles Biomedical Research Institute At Harbor-Ucla Medical Center Peptides antimicrobiens à hélices alpha-noyaux

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WO2013134734A2 (fr) * 2012-03-09 2013-09-12 Vestaron Corporation Production de peptide toxique, expression peptidique dans des plantes et combinaisons de peptides riches en cystéine
WO2017096431A1 (fr) 2015-12-09 2017-06-15 The University Of Queensland Molécules protéiques et procédés d'utilisation
WO2018126111A1 (fr) * 2016-12-30 2018-07-05 Goldstein Steven Alan Modulateurs hv1 et utilisations
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