Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-11-12
2002-07-09
Ketter, James (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S320100, C435S325000, C536S023500
Reexamination Certificate
active
06416975
ABSTRACT:
The present invention relates to nucleic acids encoding human glycine transporter type 2 (GlyT2) molecules, to proteins encoded by such nucleic acids, to methods of characterizing GlyT2-active compounds, to uses of DNA and RNA nucleotide probes directed to nucleic acids encoding the GlyT2 transporter, to uses of antisense molecules to inhibit GlyT2 expression, to uses of the GlyT2 protein to generate GlyT2-specific antibodies, and to screening methods using GlyT2-expressing cell lines, and to the field of drug discovery.
BACKGROUND
The termination of synaptic transmission in the central nervous system (CNS) involves either the enzymatic inactivation of neurotransmitters, or their uptake into pre-synaptic terminals or surrounding glial cells (Amara, S. G. and Kuhar, M. J.,
Annu. Rev. Neurosci.
16:73-93 (1998); Malandro, M. S. and Kilberg, M. S.,
Annu. Rev. Biochem.
65:305-336 (1996)). High-affinity, membrane-associated transporters typically mediate the rapid removal of neurotransmitters from the synaptic cleft, with uptake across a concentration gradient being thermodynamically coupled to transmembrane ion gradients (Kanner, B. I.,
Curr. Opin. Cell Biol.
1:735-738 (1989)).
Neurotransmitter transporters can be separated into two structurally-distinct families. One family mediates Na
30
-dependent excitatory amino acid uptake (Kanai, Y. and Hediger, M. A.,
Nature
360:467-471 (1992); Pines, G., et al.,
Nature
360:464-466 (1993); Storck, et al.,
Proc. Natl. Acad. Sci
. (USA) 89:10855-10859 (1992)). The other family contains several members involved in the Na
30
/Cl
−
-dependent transport of a host of other neurotransmitters, including GABA (Borden, L. A., et al.,
J. Biol. Chem.
267:21096-21104 (1992)); catecholamines (Pacholczyk, T., et al.,
Nature
350:350-354 (1991)); glycine (Kim, et al., Mol. Pharm. 45:608-17 (1994); Liu, Q. R., et al.,
FEBS Lett.
305:110-114 (1992a)); proline (Fremeau, R. T., et al.,
Neuron
8:915-926 (1992)) and taurine (Liu, Q. R., et al.,
Proc. Natl. Acad. Sci
. (USA) 89:12145-12149 (1992b)). The gene products for most of the latter family have recently been cloned and sequenced. These transporters demonstrate 40-50% amino acid similarity and likely exhibit structural conservation, as membrane topology analysis predicts twelve putative transmembrane regions (Guastella, J., et al.,
Science
249:1303-1306 (1990); Smith, K. E., et al.,
Neuron
8:927-935 (1992)). The high homology within these transmembrane regions has facilitated the design of degenerate primers for the cloning of additional members of the transporter superfamily using PCR-based technologies (Borowsky, B., et al.,
Neuron
10:851-863 (1993); Hoffman, B. J., et al.,
Science
254:579-580 (1991)).
Glycine is a major inhibitory neurotransmitter in the spinal cord, brainstem and retina, where it exerts its effects on the strychnine-sensitive glycine receptors (Betz, H., et al.,
Ann. N. Y. Acad. Sci.,
207:109-115 (1993); Betz, H., et al.,
Q. Rev. Biophys.
25:381-394 (1992)). In addition, glycine acts as a co-agonist with glutamate at the NMDA receptor (Kemp, J. A. and Leeson, P. D.,
Trends. Pharmacol. Sci.
14:20-25 (1993); Benveniste, M., et al.,
J. Physiol
. (London) 428:333-357 (1990)). Synaptic glycine concentrations are controlled by Na
+
/Cl
−
-dependent high-affinity transporters found at nerve-terminals and glial cells (Johnston, G. A. R. and Iverson, L. L.,
J. Neurochem.
18:1951-1961 (1971); Fedele E. and Foster A. C.,
Brain Res.
572:154-163 (1992)). Two distinct glycine transporters, GlyT1 (Smith, K. E., et al.,
Neuron
8:927-935 (1992); Liu, Q. R., et al.,
FEBS Lett.
305:110-114 (1992i); Guastella, J., et al.,
Proc. Natl. Acad. Sci.
(USA) 89:7189-7193 (1992)) and GlyT2 (Liu, Q. R., et al.,
J. Biol. Chem.
268:22802-22806 (1993)), have been isolated, and share approximately 50% identity at both the nucleotide and amino acid levels. Localization of GlyT1 and GlyT2 by in situ hybridization techniques reveals distinct patterns of expression in the CNS (Liu, Q. R., et al.,
J. Biol. Chem.
268:22802-22806 (1993); Zafra, F., et al.,
J. Neurosci.
15:3952-3969 (1995)). GlyT1 is expressed in the hippocampal and cortical regions of the brain, as well as in the spinal cord and brainstem regions. In contrast, GlyT2 is expressed primarily in the spinal cord and cerebellum, and is absent in the hippocampal and cortical regions. Based on their patterns of expression, GlyT1 is thought to co-localize with NMDA receptors, while GlyT2 expression mimics that of strychnine-sensitive glycine receptors (Jursky and Nelson,
J. Neurochem.
67:336-344 (1996); Liu, Q. R., et al.,
J. Biol. Chem.
268:22802-22806 (1993)).
The GlyT1 sequence has been determined for a number of species, including rat (Guastella, J., et al.,
Proc. Natl. Acad. Sci
. (USA) 89:7189-7193 (1992)), mouse (Liu, Q. R., et al.,
FEBS Lett.
305:110-114 (1992a)) and human (Kim, K-M., et al.,
Mol. Pharm.
45:608-17 (1994)). Three alternatively spliced forms of the human transporter have been identified (GlyT1a-c) which differ in their amino-terminal sequences (Guastella, J., et al.,
Proc. Natl. Acad. Sci
. (USA) 89:7189-7193 (1992); Liu, Q. R., et al.,
FEBS Lett.
305:110-114 (1992a); Liu, Q. R., et al.,
J. Biol. Chem.
268:22802-22806 (1993); Smith, K. E., et al.,
Neuron
8:927-935 (1992); Borowsky, B., et al.,
Neuron
10:851-863 (1993); Kim, K-M., et al.,
Mol. Pharm.
45:608-17 (1994)). The rat GlyT2 sequence has been published (Liu, Q. R., et al.,
J. Biol. Chem.
268:22802-22806 (1993)), and it also exhibits alternatively spliced forms (GlyT2a and GlyT2b) (Ponce, J., et al.,
Neurosci. Lett.
242:25-28 (1998)). Recently, two full-length clones described as pHGT2-a and pHGT2-b of human GlyT2 have been constructed (WO98/07854; PCT/US97/14637).
The precise regulation of synaptic glycine concentrations in the CNS is a very important process because glycine is involved in both excitatory and inhibitory neurotransmission (Betz, H., et al.,
Ann. N. Y. Acad. Sci.,
207:109-115 (1993); Benveniste, M., et al.,
J. Physiol
. (London) 428:333-357 (1990)). Glycine transporters are likely to be critical to this process. Compounds able to modulate glycine transporter function (i.e., that inhibit or activate glycine transporter) would be expected to provide a wide variety of therapeutic benefits. For example, glycine receptor inhibition is known to result in pain transmission (Yaksh,
Pain,
111-123, (1989)). Therefore, compounds that inhibit GlyT2 transporter activity may increase the activity of neurons having strychnine-sensitive glycine receptors via increasing synaptic levels of glycine, thus diminishing the transmission of pain-related (i.e., nociceptive) information in the spinal cord, which has been shown to be mediated by these receptors. Further, because glycine receptor malfunction is known to play a role in muscle spasticity (Becker,
FASEB J.
4:2767-2775 (1990)), compounds that inhibit GlyT2 transporter activity and lead to enhanced inhibitory glycinergic transmission through strychnine-sensitive glycine receptors in the spinal cord can be used to decrease muscle hyperactivity. Such compounds are useful in treating diseases or conditions associated with increased muscle contraction, such as muscle spasticity, myoclonus (which relates to rapid muscle spasms) and epilepsy. Spasticity that may be treated via modulation of glycine receptors is associated with epilepsy, stroke, head trauma, multiple sclerosis, spinal cord injury, dystonia, and other conditions of illness and injury of the nervous system.
SUMMARY OF THE INVENTION
The present invention provides novel human glycine transporter type 2 (GlyT2) molecules. Nucleic acids are provided comprising nucleic acid sequences that encode proteins that have glycine transport activity and that have one or more of the following amino acids: (1) serine at a position corresponding to amino acid 24, (2) tryptophan at a position corresponding to amino acid 74, (3) glycine at a position corresponding to amino acid 155, (4) aspartic acid at a
Brunden Kurt R.
Burgess Loyd H.
Gallagher Michael J.
Gliatech Inc.
Ketter James
McAndrews Held & Malloy
Schnizer Richard
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