WO1999027097A2 - Clonage d'une sous-unite alpha de canal sodique resistant a la tetrodotoxine - Google Patents
Clonage d'une sous-unite alpha de canal sodique resistant a la tetrodotoxine Download PDFInfo
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- WO1999027097A2 WO1999027097A2 PCT/US1998/024839 US9824839W WO9927097A2 WO 1999027097 A2 WO1999027097 A2 WO 1999027097A2 US 9824839 W US9824839 W US 9824839W WO 9927097 A2 WO9927097 A2 WO 9927097A2
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- 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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- This invention is generally in the field of sodium channels, and specifically is a sodium channel resistant to tetrodotoxin, and methods of screening for compounds modulating pain
- the voltage-gated sodium channels which are essential for the generation of action potentials in a wide range of excitable cells, consist of a principal ⁇ subunit and two auxiliary ⁇ -subunits (Catterall, Physiol Rev 72, S15-S48 (1992))
- Several mammalian sodium channels have been isolated from various tissues including brain, skeletal muscle, cardiac muscle, neuroendocrme cells, and dorsal root ganglia Examples include brain I, ⁇ , III (Noda et al , Nature 320, 188-192 (1986), Kayano et al , FEBS Lett 228, 187-194 (1988)), and Nach6 (Schaller et al , J Neurosci 15, 3231-3242 (1995)) from rat brain, Na s (Belcher et al , Proc Natl Acd Sci USA 92, 11034-1 1038 (1995)) from rabbit Schwann cells, its human homolog hNE- Na (Klugbauer et al
- the predicted sodium channels are strikingly similar in structure All consist of four homologous internal domains, each with six transmembrane spans (Figure 2) Most sodium channels other than Na-(1984) G show over 60% identity at the amino acid level and seem to represent a gene subfamily Na-G is only 50% identical with other sodium channels and may belong to another gene subfamily Voltage-gated sodium channels can be subdivided into three subtypes on the basis of sensitivity to tetrodotoxin (TTX), a blocker of sodium channels TTXs are obtamed from the ovaries and eggs of several species of puffer fish and certain species of California newts Chemically, tetrodotoxin is an amino perhydroquinazoline See Pharmacological Reviews.
- TTX tetrodotoxin
- TTX-sensitive brain and skeletal muscle sodium channels are blocked by nanomolar level (1-5 nM) of TTX, and the TTX-insensitive cardiac sodium channels are blocked by micromolar (0 3-1 0 ⁇ M) TTX, while the TTX-resistant channels are resistant to very high concentrations (30-100 ⁇ M) of TTX (Frehn et al , Eur J of Pharm 122 245-250 (1986), White et al , Neuron 1 1, 1037-1047 (1993))
- sodium channels, especially TTX- resistant channels might be responsible for the excitability of nociceptors and underlie pain and tenderness associated with tissue injury and inflammation (Devor et al , Neurosci 13, 1976-1992 (1993), Waxman et al , J Neurophys 72, 466-470 (1994), Jeftinija et al , Brain Res 639, 125-134 (1994))
- TTX-resistant channels might be responsible for the excitability of noc
- TTX-resistant sodium channels might play an important role in the regulation of nociceptors.
- the nodose ganglia contains most of the sensory cell bodies of the vagus neuron. These sensory neurons are concerned with visceral sensation from the heart, larynx, lungs and alimentary tract from the pharynx to the transverse colon.
- Previous reports showed that the TTX-resistant sodium channel also exists in group C sensory neurons from nodose ganglia (Bossu et al., Neurosci. Lett 51, 241-246 (1984)). Therefore, tetrodotoxin-resistant sodium channels may play an important role in visceral sensation and visceral pain regulation.
- Pharmacological therapies designed to specifically block tetrodotoxin- resistant sodium channels might be a potential treatment for pain disorders. It is therefore an object of the present invention to provide another TTX-resistant sodium channel, and means for screening for drugs altering its activity.
- a full length cDNA includes an open-reading frame of 5,886 nucleotides (SEQ ID NO 1), which encodes a protein of 1,962 amino acids (SEQ LD NO.2).
- SEQ ID NO 1 an open-reading frame of 5,886 nucleotides
- SEQ LD NO.2 1,962 amino acids
- RT-PCR reverse transcription-polymerase chain reaction
- the deduced amino acid sequence of NaNG (SEQ ID NO:2) includes 1,962 amino acids with a calculated molecular weight of 220.7 kd. It displays the features common to all sodium channels with four large homologous domains, each containing six potential membrane spanning helical segments (S1-S6) including a positively charged segment (S4). The regions of greatest resemblance to other sodium channels are displayed at L3, S4, SSI and SS2.
- the linker between III6 and IV1 is 98.4% identical with SNS/PN3 and the rat cardiac channel, consistent with the fact that this region might be essential for inactivation
- the S4 segment contains positively charged amino acids and presumably acts as the voltage sensor SS 1 and SS2, the extracellular linker of S5-S6, are components of the pore structure and thus might play an important role in ion conductance and selectivity
- the two other large intracellular domains, Ll and L2 are much more divergent, and thus may confer unique functions and pharmacology Gin-related glycosylation sites are also present in NaNG
- Most of the ten potential glycosylation sites cluster in the extracellular linkers of S5 and S6 (four between D-I S5 and IS6, three between D-III S5 and D-HI S6) NaNG does not contain the PKA site present in lnterdomain II-III of SNS/PN3, but contains a unique C-terminal site 1958 Thr, which might be very important in channel regulation
- NaNG represents a unique target for compounds involved in regulations of pain
- Compounds can be screened for binding in vitro, alterations of sodium passage through the channel in vitro, or in vivo, using standard techniques to screen for local anesthetics and "pain killers"
- Figure 1 is a schematic drawing of the strategy for cloning the full- length NaNG (SEQ ID NO 1)
- Figure 2 is a functional map of a sodium channel ⁇ -subunit linearly illustrating the four homologous domains (D-I, II, II and IV), the membrane spanning regions (represented by cylinders S1-S6), pore forming segments (SSI and SS2) and the intracellular linkers (Ll-3).
- Figures 3 A-3G are the nucleotide sequence of NaNG (SEQ ID NO: l).
- Figures 4A-4D are the deduced amino acid sequence (SEQ ED NO:2) and primary structure of NaNG according to the alignment with cloned sodium channels. The membrane spanning domains, and pore-forming segments SSI and SS2 are marked. *-TTX-resistance site, V-potential N- linked glycosylation site, ⁇ -potential cAMP-dependent protein kinase (PKA) phosphorylation site, --potential protein kinase C phosphorylation site.
- PKA protein kinase C phosphorylation site
- Figures 5 A and 5B are a partial amino acid sequence between IS5 and IS6 of NaNG (amino acids 264-385 of SEQ ED NO:2) and the alignment of this fragment with rat SNS (SEQ ID NO: 3).
- pFL full-length cDNA clone pFL (Nt 237-6524) including coding and parts of the 5', 3 -noncoding region was constructed.
- pFL was completely sequenced and compared to FI, F2 and F3 and the additional fragments F4 and F5 (described in Example 4).
- Several sequence discrepancies were eliminated by additional RT-PCR, such as A ⁇ T change at Nt 3031 of SEQ ID NO: l .
- the analysis resulted in a full length cDNA of 6,726 nucleotides (SEQ ID NO: l), consisting of 258 base pair (bp) of 5' noncoding region, an opening reading frame of 5,886 bp and
- Homo logs of the disclosed dog TTX-resistant sodium channel are also expressed in other mammalian nodose ganglion tissue, in particular, human tissue.
- the dog sodium channel cDNA (SEQ ID NO. 1) can be used as a probe to detect and isolate the homologue TTX-resistant sodium channel in human nodose ganglion tissue. Standard techniques can be used.
- the human homologue of the dog TTX-resistant NaNG can be cloned using a human nodose ganglion cDNA library. Human nodose ganglion are obtained at autopsy. The frozen tissue is homogenized and the RNA extracted with guanidine isothiocyanate (Chirgwin, et al. Biochemistry 18:5294-5299, 1979).
- RNA is size-fractionated on a sucrose gradient to enrich for large mRNAs because the sodium channel ⁇ -subunits are encoded by large (7-1 1 kb) transcripts.
- Double-stranded cDNA is prepared using the Superscript Choice cDNA kit (GEBCO BRL) with either oligo (dT) or random hexamer primers. EcoRI adapters are ligated onto the double- stranded cDNA which is then phosphorylated.
- the cDNA library is constructed by ligating the double-stranded cDNA into the bacteriophage-
- Phage are plated out on 150 mm plates on a lawn of XLI-Blue MRF' bacteria (Stratagene) and plaque replicas are made on Hybond N nylon membranes (Amersham). Filters are hybridized to a dog NaNG cDNA or cRNA probe by standard procedures and detected by autoradiography or chemiluminescence. The signal produced by the dog NaNG probe hybridizing to positive human clones at high stringency should by stronger than obtained with dog brain sodium channel probes hybridizing to these clones. Positive plaques are further purified by limiting dilution and re- screened by hybridization or PCR. Restriction mapping and polymerase chain reaction will identify overlapping clones that can be assembled by standard techniques into the full-length human homologue of dog NaNG.
- the human clone can be expressed by injecting cRNA transcribed in vitro from the full-length cDNA clone into Xenopus oocytes, or by transfecting a mammalian cell line with a vector containing the cDNA linked to a suitable promoter.
- the deduced amino acid sequence of NaNG (SEQ ID NO: 2) contains 1,962 amino acids with a calculated molecular weight of 220.7 kd. It displays the features common to all sodium channels with four large homologous domains, each containing six potential membrane spanning helical segments (S1-S6) including a positively charged segment (S4) illustrated in Figures 2 and 4A-4D.
- S1-S6 potential membrane spanning helical segments
- S4 positively charged segment
- Table 1 The similarities between NaNG and other known sodium channels are demonstrated in Table 1. At the amino acid level, it most closely (82.3%) resembles SNS/PN3 and least (46.5%) resembles the hNA v 2.1 (Akopian et al., Nature 379, 257-262 (1996); Sangameswaran et al., J. Biol. Chem.
- Brain III 60.1 50.4 66.9 39.9 69.3 37.3 74.5 85.5 70.3 50.4
- Percent identities are derived from comparison of NaNG with SNS/PN3 (Akopian et al., Nature 379, 257-262 (1996); Sangameswaran et al., J. Biol. Chem. 271, 5993-5986 (1996)), cardiac (Gellens et al., Proc. Natl. Acad. Sci. USA 89, 554-558 (1992)), Brain I, Brain II (Noda et al.,
- hNa v 2.1 (George et al., Ann Neurol 31, 131-7 (1992) and George et al , Proc Natl Acad Sci USA 89, 4893-4897 (1992))
- Na is from rabbit
- hNa v 2 1 is from human
- others are from rat Total overall sequence, N N- terminus, IS-IVS membrane-spanning repeats, L1-L3 intracellular linkers, C C-terminus
- Ll and L2 are much more divergent, and thus may confer unique functions and pharmacology
- NaNG shares the TTX-binding site with the TTX-resistant SNS/PN3 In TTX-sensitive channels, a Phe or Tyr is involved in high affinity binding to TTX In TTX-insensitive sodium channels, this residue is replaced with a hydrophilic Cys, while in NaNG, it is a more hydrophilic Ser, which is exactly the same as SNS PN3 Based on these facts, NaNG is believed to be a TTX-resistant sodium channel
- NaNG is the first sodium channel obtained from dog tissue
- the homologs from different mammalian species are highly conserved.
- rat and mouse SNS/PN3 are 95.1% identical at the amino acid level (mouse SNS- Genbank accession number Y09108); human and rat cardiac sodium channels are 94.0% identical (Rogart et al., Proc. Natl. Acad. Sci. USA 86, 8170-8174 (1989); Gellens et al., Proc. Natl. Acad. Sci.
- the linker between IS5 and IS6 is the pore-forming region, which is very important in defining channel characteristics.
- Oligo 410 and 411 several partial clones containing the linker between IS5 and IS6 have also been obtained ( Figures 5A-5B).
- the dog partial clones of brain I, brain III, NaCh ⁇ and NA-G are all above 92% identical with their rat counterparts.
- Dog Na-G partial clone and human Na-G are 86.7% identical, while in the linker between IS5 and IS6, NaNG and rat SNS/PN3 are only 72.9% identical. Therefore it is believed that NaNG is a novel sodium channel, possibly belonging to the same gene subfamily of the TTX-resistant sodium channels at SNS/PN3.
- Coupled transcription and translation with the full length clone pFL produced a protein of approximately 220 kd, which is of the same size as the
- RNA were blotted and hybridized with a labeled F2 fragment.
- a 7.0 kb transcript was revealed only in nodose ganglia, not in cortex, hippocampus, cerebellum, liver, heart or skeletal muscle.
- NaNG distribution in multiple dog tissues S6 and AS7 were used to amplify a unique 202 bp fragment from the NaNG transcript (Nt 1589-1790). This fragment was run on a gel with a 50-2,000 bp DNA Marker and pFL as a positive control. Amplification products were obtained only from nodose ganglia.
- the cDNA predicts a protein encoding a voltage-gated sodium channel (NaNG) from dog nodose ganglia that exhibits features of known
- the restrictive tissue distribution of NaNG makes it possible for NaNG to play an important role in visceral sensation or visceral pain regulation.
- the restrictive tissue distribution has value for developing therapeutics targeted for a specific tissue that will not inhibit sodium channels in a wide range of tissues.
- the detection of NaNG mRNA in nodose ganglia suggests that NaNG may conduct TTX-resistant sodium currents in this sensory ganglia.
- the distribution of NaNG indicates it is strictly regulated at a transcriptional level.
- Posttranslational modification such as PKA PKC- mediated phosphorylation and N-linked glycosylation also might be involved in NaNG modulation.
- Methods for screening for drugs that bind to NaNG to alter activity in vitro include those that selectively bind NaNG and modulate NaNG function. Binding can be determined using standard techniques. Modulation of NaNG, as demonstrated by compounds showing potent in vitro affinity for NaNG, make the compounds useful for treating pain and tenderness associated with tissue injury and inflammation.
- Examples of analytical methods to determine the binding for each ligand include exposing NaNG to a radiolabeled ligand and increasing amounts of test ligand. The amount of radioactivity bound to NaNG will decrease in the presence of test compounds which compete for the binding site for the radiolabeled ligand.
- Methods for screening for drugs that bind to NaNG to alter activity in vivo include testing anesthetics in animal studies. For example, animals may be anesthetized and a nerve block may be performed by injecting a drug to block one limb, while the opposite limb is used as a control.
- ISA/EP effectiveness of the nerve block is measured at various time points. Standard parameters to be assessed include nociceptive blocks (heat), tactile placing responses (a prone leg will be pulled back), hopping, and extensor postural thrust (the maximum weight that an animal will bear without an ankle touching a balance is measured). Examples
- a first strand cDNA was synthesized using random hexamer primers.
- Degenerate oligonucleotides 410, 411 (shown in Table 2) were used to amplify sodium channel sequences by PCR (Gene Amp PCR system 2400, Perkin Elmer). Each reaction consisted of 200 ⁇ M dNTPS, 1 ⁇ M each primer and 2.5 units of Expand Long Template System polymerases (Boehringer Mannheim). Thermocycling parameter included 30 cycles of 94°C 10 sec, 45°C 30 sec and 68°C 1 min. RT-PCR products were digested with Baml/EcoRI, cloned into pGEM 3 and sequenced. A unique sequence different from all known sodium channels was identified and called NaNG.
- Nucleotide sequence, position in NaNG sequence and corresponding am o acids are given for each primer API and AP2 are based on adaptor sequence, Ol ⁇ go410 01 ⁇ go411 and AS3 are degenerate pnmers designed from conserved sequences of known sodium channels, nucleotide code follows convention of the International Union of Biochemistry and Molecular Biology, S3 and AS6 are in the nontranslational region of NaNG The incorporated restriction sites are underlined *S-sense AS-antisense
- HMLV reverse transcriptase New England Biolabs
- HMLV is more efficient for synthesizing cDNA of large size.
- AS 1 and AS2 primers were based on the unique sequence and designed for 5'-RACE. Nested PCR was performed with AS1/AP1 for the primary round and AS2/AP2 for the secondary round PCR.
- PCR parameters were: 30 cycles of 10 seconds of denaturing at 93 °C, 20 sec of annealing at 60°C and 2 min of extension at 68°C.
- the PCR product was cloned, completely sequenced and called FI.
- Example 3 3'-RACE and cloning of 3'-cDNA end cDNA 3 '-ends were obtained by two successive rounds of RACE. In the first step, degenerate primer AS3 (targeting a conserved domain) was used to facilitate walking in the 3 '-end direction. Nested PCR (SI/API, S2/AS3) product-fragment F2 was cloned and its sequence was used to design a 3' walking primer S5.
- Oligos around this site were designed to generate two overlapping fragments-F4 (AS4/AP1, AS5/S3) and F5 (S I/API , S4/AP6), which covered coding region and parts of 5' and 3' noncoding regions F4 and F5 were cloned respectively in pCI vector (Promega) and subsequently a full-length cDNA (pFL) was cloned between Xbal and NotI sites of the pCI by Kpnl site-mediated subcloning DNA sequences and predicted amino acid sequences were analyzed with programs of the GCG package (Genetics Computer Group, Inc )
- Example 5 In vitro transcription and translation assay of NaNG The full-length clone pFL was assayed with the TNT R Coupled Reticulocyte Lysate System (Promega) as described in the manual, using 35 S- methionine (Amersham) incorporation pCI vector (Promega) was included as negative control and Luciferase DNA as a positive control Translation products were analyzed by SDS-PAGE and autoradiography Example 6.
- Prehyb ⁇ dization and hybridization were carried out as described (Sambrook et al , 1989), washed one time with lxSSC, 0 1% SDS, 30 min at 42°C, washed two times with 0 lxSSC, 0 1% SDS, 30 mm at 65°C, then exposed to Kodak XAR film with an intensifying screen at -70°C for 5 days
- First strand cDNA was synthesized from total RNA isolated from the tissues described above The cDNA was subjected to PCR with primers S6 and AS7, which were designed to amplify a unique 202 bp fragment from the NaNG transcript (nt 1589 to 1790) Clone pFL was included as a positive control
- NaNG has Gen Bank accession number U60590
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AU14661/99A AU1466199A (en) | 1997-11-20 | 1998-11-20 | Cloning of a tetrodotoxin-resistant sodium channel alpha-subunit |
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US6621797P | 1997-11-20 | 1997-11-20 | |
US60/066,217 | 1997-11-20 |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6184349B1 (en) | 1995-10-11 | 2001-02-06 | Syntex (Usa) Inc. | Cloned peripheral nerve, tetrodotoxin-resistant sodium channel α-subunit |
CN104311569A (zh) * | 2014-09-05 | 2015-01-28 | 无锡科奥美萃生物科技有限公司 | 一种提取和初步纯化河豚毒素的方法 |
Citations (1)
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WO1997001577A1 (fr) * | 1995-06-28 | 1997-01-16 | University College London | Canal ionique |
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- 1998-11-20 WO PCT/US1998/024839 patent/WO1999027097A2/fr active Application Filing
- 1998-11-20 AU AU14661/99A patent/AU1466199A/en not_active Abandoned
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WO1997001577A1 (fr) * | 1995-06-28 | 1997-01-16 | University College London | Canal ionique |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6184349B1 (en) | 1995-10-11 | 2001-02-06 | Syntex (Usa) Inc. | Cloned peripheral nerve, tetrodotoxin-resistant sodium channel α-subunit |
US6479259B1 (en) | 1995-10-11 | 2002-11-12 | Syntex (U.S.A.) Llc | Human peripheral nerve, tetrodotoxin-resistant sodium channel α-subunit polypeptide |
US7122325B2 (en) | 1995-10-11 | 2006-10-17 | Syntex (U.S.A.) Llc | Method of modulating tetrotodoxin-resistant sodium channel |
CN104311569A (zh) * | 2014-09-05 | 2015-01-28 | 无锡科奥美萃生物科技有限公司 | 一种提取和初步纯化河豚毒素的方法 |
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AU1466199A (en) | 1999-06-15 |
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