WO2001068879A2

WO2001068879A2 - Human 1-aminocyclopropane-carboxylate synthase

Info

Publication number: WO2001068879A2
Application number: PCT/EP2001/002857
Authority: WO
Inventors: Shyam Ramakrishnan
Original assignee: Bayer Aktiengesellschaft
Priority date: 2000-03-14
Filing date: 2001-03-14
Publication date: 2001-09-20
Also published as: WO2001068879A3; AU2001242457A1

Abstract

Reagents which regulate human 1-aminocyclopropane-carboxylate (ACPC) synthase activity and reagents which bind to human ACPC synthase gene products can be used to regulate brain function. Such regulation is particularly useful for treating ischemia and trauma of the brain and spinal cord as well as neurodegenerative diseases and other diseases related to disturbances of N-methyl-D-aspartate (NMDA) receptor activity.

Description

REGULATION OF HUMAN 1-AMINOCYCLOPROPANE-CARBOXYLATE SYNTHASE

TECHNICAL FIELD OF THE INVENTION

The invention relates to the regulation of nerve function. More particularly, the invention relates to the regulation of human NMD A receptor activity and its use in the treatment of neurodegeneration.

BACKGROUND OF THE INVENTION

Extensive loss of neurons occurs after ischemic insult in brain or spinal cord. Ischemia produces anoxic depolarization, which leads to the release of glutamate and consequent activation of N-methyl-D-aspartate (NMD A) receptors. NMD A receptors are ligand-gated ion channels which respond to glutamate by allowing calcium ions to enter nerve cells (McBain, CJ. and Mayer, M.L., Physiol. Rev. 74: 723 (1994)). Excessive activation of NMD A receptors by glutamate can lead to the elevation of intracellular calcium to toxic levels, resulting in ischemic injury.

Electrophysiological studies have shown that 1 -aminocyclopropane carboxylic acid (ACPC) is a partial agonist for NMDA receptors and possesses high intrinsic activity (Priestly, T. and Kemp, J.A., Mol. Pharmacol. 46:1191 (1994)). ACPC acts both as an agonist at the glycine binding site of the NMDA receptor and as an antagonist at the glutamate binding site (Nahum-Levy, R., Fossom, L.H., Skolnick, P., and

Benveniste, M., Mol. Pharmacol. 56:1207 (1999)). Due to its antagonism of glutamate, ACPC has been found to exert a neuroprotective effect in vivo (Long and Skolnick, 1994). rendering it useful in the treatment of stroke and other ischemic conditions, hypoglycemia, traumatic injury of the brain or spinal cord, and neurodegenerative diseases such as Parkinson's disease, Huntington's disease, and dementia (Robinson, M, and Coyle, J., FASEB J.1 :446 (1987))). Antagonism of the neuroexcitatory function of glutamate makes ACPC useful in the treatment of epilepsy (Robinson and Coyle, supra). ACPC can aid in the treatment of depression and anxiety (Przegalinski, E., Tatarczynska, E., Klodzinska, A., and Chojnacka- Wojcik, E., Pharmcol. Biochem. Behav. 64:461 (1999)), and drug or alcohol addiction (Stromberg, M.F., Volpicelli, J.R., O'Brien, C.P., and Mackler, S.A.,

Pharmacol. Biochem. Behav. 64:585 (1999)). ACPC also facilitates spatial learning and memory (Popik, P. and Rygielska, Z., J. Physiol. Pharmacol. 50:139 (1999)).

The clinical utility of NMDA receptor antagonists, including the uses outlined above, is frequently limited by the undesirable side effects of this class of drugs. NMDA antagonists are icnown to cause muscle relaxation, ataxia, amnesia, and psycho- mimetic effects (Przegalinski, supra). ACPC, however, is devoid of these effects (Papp, M., and Moryl, E., Eur. J. Pharmacol.316:145 (1996)) and therefore offers advantages over other NMDA receptor antagonists. Furthermore, chronic administration of ACPC, which is not possible with other NMDA receptor antagonists due to undesirable side effects, may offer superior protection from ischemic damage than acute administration. The sustained effect of ACPC is thought to involve selective alteration of the mRNA expression levels of certain subunits of the NMDA receptor (see, e.g., Bovetto, S., Boyer, P.A., Skolnick, P., and Fossom, L.H., J. Pharmacol. Exp. Therap. 283:1503 (1997)). Thus, there is a need for methods to administer ACPC and drugs to modulate ACPC synthase activity so as to treat neurodegenerative conditions and regulate neuronal function in a variety of diseases.

SUMMARY OF THE INVENTION

It is an object of the invention to provide reagents and methods for regulating excitatory neuro transmission. It is another object of the invention to provide reagents and methods for preventing neurodegeneration. These and other objects of the invention are provided by one or more ofthe embodiments described below. One embodiment of the invention is a ACPC synthase polypeptide comprising an amino acid sequence selected from the group consisting of:

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 2;

the amino acid sequence shown in SEQ ID NO: 2;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 4;

the amino acid sequence shown in SEQ ID NO:4;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 6;

the amino acid sequence shown in SEQ ID NO: 6;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 8;

the amino acid sequence shown in SEQ ID NO: 8;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 10;

the amino acid sequence shown in SEQ ID NO: 10;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 12; the amino acid sequence shown in SEQ ID NO: 12;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 14;

the amino acid sequence shown in SEQ ID NO: 14;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 16;

the amino acid sequence shown in SEQ ID NO: 16;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 17; and

the amino acid sequence shown in SEQ ID NO: 17.

Yet another embodiment of the invention is a method of screening for agents which decrease the activity of an ACPC synthase. A test compound is contacted with a ACPC synthase polypeptide comprising an amino acid sequence selected from the group consisting of:

the amino acid sequence shown in SEQ ID NO: 2;

the amino acid sequence shown in SEQ ID NO:4; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 6;

the amino acid sequence shown in SEQ ID NO: 6;

mino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 8;

the amino acid sequence shown in SEQ ID NO: 8;

the amino acid sequence shown in SEQ ID NO: 10;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 12;

the amino acid sequence shown in SEQ ID NO: 12;

the amino acid sequence shown in SEQ ID NO: 14;

the amino acid sequence shown in SEQ ID NO: 16; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 17; and

the amino acid sequence shown in SEQ ID NO: 17.

Binding between the test compound and the ACPC synthase polypeptide is detected. A test compound which binds to the ACPC synthase polypeptide is thereby identified as a potential agent for decreasing the activity of an ACPC synthase. Another embodiment of the invention is a method of screening for agents which decrease the activity of an ACPC synthase. A test compound is contacted with a polynucleotide encoding an ACPC synthase polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1;

the nucleotide sequence shown in SEQ ID NO: 1;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 3;

the nucleotide sequence shown in SEQ ID NO: 3;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 5;

the nucleotide sequence shown in SEQ ID NO:5;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 7; the nucleotide sequence shown in SEQ ID NO: 7;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 9;

the nucleotide sequence shown in SEQ ID NO: 9;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 11;

the nucleotide sequence shown in SEQ ID NO:l 1;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 13;

the nucleotide sequence shown in SEQ ID NO: 13;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 15; and

the nucleotide sequence shown in SEQ ID NO: 15.

Binding of the test compound to the polynucleotide is detected. A test compound which binds to the polynucleotide is identified as a potential agent for decreasing the activity of an ACPC synthase. The agent can work by decreasing the amount of the

ACPC synthase through interacting with the ACPC synthase mRNA.

Another embodiment of the invention is a method of screening for agents which regulate the activity of an ACPC synthase. A test compound is contacted with a ACPC synthase polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 2;

the amino acid sequence shown in SEQ ID NO: 2;

the amino acid sequence shown in SEQ ID NO:4;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 6; and

the amino acid sequence shown in SEQ ID NO: 6.

the amino acid sequence shown in SEQ ID NO: 8;

the amino acid sequence shown in SEQ ID NO : 10;

the amino acid sequence shown in SEQ ID NO: 12; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 14;

the amino acid sequence shown in SEQ ID NO: 14;

the amino acid sequence shown in SEQ ID NO: 16;

the amino acid sequence shown in SEQ ID NO: 17.

An ACPC synthase activity of the polypeptide is detected. A test compound which increases ACPC synthase activity of the polypeptide relative to ACPC synthase activity in the absence of the test compound is thereby identified as a potential agent for increasing the activity of an ACPC synthase. A test compound which decreases ACPC synthase activity of the polypeptide relative to ACPC synthase activity in the absence of the test compound is thereby identified as a potential agent for decreasing the activity of an ACPC synthase.

Even another embodiment ofthe invention is a method of screening for agents which decrease the activity of an ACPC synthase. A test compound is contacted with a

ACPC synthase product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of:

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1 ; the nucleotide sequence shown in SEQ ID NO: 1;

the nucleotide sequence shown in SEQ ID NO: 3;

the nucleotide sequence shown in SEQ ID NO:5;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 7;

the nucleotide sequence shown in SEQ ID NO: 7;

the nucleotide sequence shown in SEQ ID NO: 9;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 11 ;

the nucleotide sequence shown in SEQ ID NO: 11;

the nucleotide sequence shown in SEQ ID NO: 13; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 15; and

the nucleotide sequence shown in SEQ ID NO: 15.

Binding of the test compound to the ACPC synthase product is detected. A test compound which binds to the ACPC synthase product is thereby identified as a potential agent for decreasing the activity of an ACPC synthase.

Still another embodiment of the invention is a method of reducing the activity of an ACPC synthase. A cell is contacted with a reagent which specifically binds to a polynucleotide encoding a ACPC synthase polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:

the nucleotide sequence shown in SEQ ID NO: 1 ;

the nucleotide sequence shown in SEQ ID NO: 3;

the nucleotide sequence shown in SEQ ID NO:5; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 7;

the nucleotide sequence shown in SEQ ID NO: 7;

the nucleotide sequence shown in SEQ ID NO: 9;

the nucleotide sequence shown in SEQ ID NO: 11 ;

the nucleotide sequence shown in SEQ ID NO: 13;

the nucleotide sequence shown in SEQ ID NO: 15.

ACPC synthase activity in the cell is thereby decreased.

The invention thus provides reagents and methods for regulating ACPC synthase activity. Such reagents and methods can be used inter alia, to regulate NMDA receptor activity and to prevent nerve damage and neuronal death following ischemia or in a variety of neurodegenerative conditions. BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1A-1B Alignment of human ACPC synthase (SEQ ID NO: 14) with the protein assigned GenBank Accession Number AF 108420 (SEQ ID NO: 17).

Fig. 2 DNA-sequence encoding an ACPC synthase polypeptide.

Fig. 3 Amino acid sequence ofthe ACPC synthase polypeptide of Fig.2.

Fig. 4 DNA-sequence encoding an ACPC synthase polypeptide.

Fig. 5 Amino acid sequence ofthe ACPC synthase polypeptide of Fig.4.

Fig. 6 DNA-sequence encoding an ACPC synthase polypeptide.

Fig. 7 Amino acid sequence ofthe ACPC synthase polypeptide of Fig.6.

Fig. 8 DNA-sequence encoding an ACPC synthase polypeptide.

Fig. 9 Amino acid sequence ofthe ACPC synthase polypeptide of Fig.8.

Fig. 10 DNA-sequence encoding an ACPC synthase polypeptide.

Fig. 11 Amino acid sequence ofthe ACPC synthase polypeptide of Fig.10.

Fig. 12 DNA-sequence encoding an ACPC synthase polypeptide.

Fig. 13 Amino acid sequence of the ACPC synthase polypeptide of Fig.12. Fig. 14 DNA-sequence encoding an ACPC synthase polypeptide.

Fig. 15 Amino acid sequence of the ACPC synthase polypeptide of Fig.14.

Fig. 16 DNA-sequence encoding an ACPC synthase polypeptide.

Fig. 17 Amino acid sequence ofthe ACPC synthase polypeptide of Fig.16.

Fig. 18 Amino acid sequence of an ACPC synthase polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an isolated polynucleotide encoding an ACPC synthase polypeptide and being selected from the group consisting of:

a) a polynucleotide encoding an ACPC synthase polypeptide comprising an amino acid sequence selected from the group consisting of:

amino acid sequences which are at least about 50% identical to

the amino acid sequence shown in SEQ ID NO: 2;

amino acid sequences which are at least about 50% identical to

the amino acid sequence shown in SEQ ID NO: 4;

the amino acid sequence shown in SEQ ID NO:4; amino acid sequences which are at least about 50% identical to

the amino acid sequence shown in SEQ ID NO: 6;

amino acid sequences which are at least about 50% identical to

the amino acid sequence shown in SEQ ID NO: 8;

amino acid sequences which are at least about 50% identical to

the amino acid sequence shown in SEQ ID NO: 10;

amino acid sequences which are at least about 50% identical to

the amino acid sequence shown in SEQ ID NO: 12;

amino acid sequences which are at least about 50% identical to

the amino acid sequence shown in SEQ ID NO: 14;

the amino acid sequence shown in SEQ ID NO: 14; amino acid sequences which are at least about 50% identical to

the amino acid sequence shown in SEQ ID NO: 16;

amino acid sequences which are at least about 50% identical to

the amino acid sequence shown in SEQ ID NO: 17; and

the amino acid sequence shown in SEQ ID NO: 17.

b) a polynucleotide comprising the sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 or 15;

c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b);

d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code; and

a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d).

Furthermore it has been discovered by the present applicant that the activity of an ACPC synthase, particularly a human ACPC synthase, can be modified to regulate excitatory neurotransmission via the NMDA receptor. Human ACPC synthase produces 1-aminocyclopropane carboxylic acid (ACPC). ACPC synthase was previously known to exist in plants. ACPC is important as a precursor of ethylene.

ACPC synthase (S-adenosyl-L-methionine methyl thioadenosine- lyase, EC4.4.1.14; also called ACC synthase) (Kionka C, et al., (1984) Planta 162: 226-235) is a cytosolic enzyme that converts S-adenosylmethionine to ACPC and methyl- thioadenosine. Plant ACPC synthase also uses pyridoxal phosphate as a cofactor. The reaction catalyzed by ACPC synthase is the rate-limiting step of ethylene synthesis in plants. Ethylene is important in plant growth and development (Abeles,

F. B. et al. (1992), In: Ethylene in Plant Biology, eds. Abeles, F. B. et al., Academic Press, New York, pp 285-291 and 1-13; Yang, S. F. et al. (1984), Annu.. Rev. Plant Physiol.: 35, 155-189). Ethylene production in plants is also associated with trauma induced by mechanical wounding, chemicals, stress, and disease. ACPC synthase is encoded by a multigene family in plants, including every plant species examined

(Zarembinski, T.I., and Theologis, A. (1994) Plant Mol. Biol. 26:1579).

Messenger RNA sequences encoding ACPC synthase are now known to be expressed in human cells. The cDNA sequences shown as SEQ ID NOS:l, 5, 7, and 9 and complement of SEQ ID NO:3 are now known to be expressed in human cells.. The respective amino acid sequences encoded by those mRNA sequences are shown as SEQ ID NOS: 2, 4, 6, 8, and 10. The discovery of ACPC synthase in human cells indicates that ACPC, which has been administered as a drug in humans, is also produced within the human body. Endogenous production of ACPC can now be modified using the reagents and methods ofthe invention.

Human ACPC synthase can be used to develop treatments for various diseases, to develop diagnostic assays for these diseases, and to provide new tools for basic research especially in the fields of medicine and biology. Specifically, the present invention can be used to develop new drugs to activate or inhibit ACPC synthase, as well as to modulate the activity of NMDA receptors. Human ACPC synthase and regulators of human ACPC synthase thus can provide treatments for stroke and other ischemic conditions. Human ACPC synthase also can be used to treat nerve damage resulting from hypoglycemia or from traumatic injury ofthe brain or spinal cord. Furthermore, human ACPC synthase can be used to treat neurodegenerative diseases such as Parkinson's disease, Huntington' s disease, and dementia. ACPC Synthase Polypeptides

Human ACPC synthase polypeptides according to the invention comprise an amino acid sequence as shown in SEQ ID NOS:2, 4, 6, 8, or 10, a portion of one of those amino acid sequences, or a biologically active variant of an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, or 10, as defined below. The asterisks represent the positions of stop codons introduced by sequencing errors into the ESTs encoding these amino acid sequences. Thus, an ACPC synthase polypeptide can be a portion of an ACPC synthase molecule, a full-length ACPC synthase molecule, or a fusion protein comprising all or a portion of an ACPC synthase molecule. Most preferably, an ACPC synthase polypeptide has an ACPC synthase activity. ACPC synthase activity can be measured, inter alia, as described in Example 2.

Some portions ofthe amino acid sequences shown in SEQ ID NOS:2, 4, 6, 8, and 10 which are of particular interest are the portion of SEQ ID NO:2 encoded by nucleotide residues 183 to 287 of SEQ ID NO:l; the portion of SEQ ID NO:4 encoded by the complement of nucleotide residues 242 to 340 of SEQ ID NO:3; the portion of SEQ ID NO:6 encoded by nucleotide residues 276 to 443 of SEQ ID NO:5; the portion of SEQ ID NO:8 encoded by nucleotide residues 1 to 69 of SEQ ID NO:7; and the portion of SEQ ID NO: 10 encoded by nucleotide residues 2 to 70 of SEQ ID NO:9.

Biologically Active Variants

ACPC synthase variants which are biologically active, i.e., retain an ACPC synthase activity, also are ACPC synthase polypeptides. Preferably, naturally or non-naturally occurring ACPC synthase variants have amino acid sequences which are at least about 50, preferably about 75, 90, 96, or 98% identical to an amino acid sequence shown in SEQ ID NOS:2. 4. 6. 8. or 10. Percent identity between a putative ACPC synthase variant and an amino acid sequence of SEQ ID NO:2, 4. 6, 8, or 10 is determined using the Blast2 alignment program. Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of an ACPC synthase polypeptide can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active ACPC synthase polypeptide can readily be determined by assaying for ACPC synthase activity, as described, for example, in Example 2.

Fusion Proteins

Fusion proteins can comprise at least 5, 6, 8, 10, 25, or 50 or more contiguous amino acids of an amino acid sequence shown in SEQ ID NO:2, 4, 6, 8, or 10. Fusion proteins are useful for generating antibodies against ACPC synthase amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of an ACPC synthase polypeptide. Protein affinity chromatography or library-based assays for protein- protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.

An ACPC synthase fusion protein comprises two protein segments fused together by means of a peptide bond. The first protein segment comprises at least 5, 6, 8, 10, 25, or 50 or more contiguous amino acids of an ACPC synthase polypeptide. Contiguous amino acids for use in a fusion protein can be selected from an amino acid sequence shown in SEQ ID NO:2, 4, 6, 8, or 10 or from a biologically active variant of those sequences, such as those described above. The first protein segment also can comprise full-length ACPC synthase.

The second protein segment can be a full-length protein or a protein fragment or polypeptide. Proteins commonly used in fusion protein construction include - galactosidase, β-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV- G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4

DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the ACPC synthase polypeptide-encoding sequence and the heterologous protein sequence, so that the ACPC synthase polypeptide can be cleaved and purified away from the heterologous moiety.

A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two protein segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NO. l, 5, 7, or 9 or the complement of SEQ ID NO:3 in proper reading frame with nucleotides encoding the second protein segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA),

CLONTECH (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

Identification of Species Homologs

Species homologs of human ACPC synthase can be obtained using ACPC synthase polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of ACPC synthase, and expressing the cDNAs as is known in the art.

ACPC Synthase Polynucleotides

An ACPC synthase polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for an ACPC synthase polypeptide. Partial nucleotide sequences encoding ACPC synthase polypeptides are shown in SEQ ID NOS:l, 5, 7, and 9; the complement of a partial nucleotide sequence encoding an ACPC synthase polypeptide is shown in SEQ ID NO:3.

Degenerate nucleotide sequences encoding human ACPC synthase polypeptides, as well as homologous nucleotide sequences which are at least about 50, preferably about 75, 90, 96, or 98% identical to the complements of the nucleotide sequences shown in SEQ ID NO:l, 3, 5, 7, or 9, also are ACPC synthase polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affϊne gap search with a gap open penalty of -12 and a gap extension penalty of -2. Complementary DNA (cDNA) molecules, species homologs, and variants of ACPC synthase polynucleotides which encode biologically active ACPC synthase polypeptides also are ACPC synthase polynucleotides. Identification of Variants and Homologs of ACPC Synthase Polynucleotides

Variants and homologs of the ACPC synthase polynucleotides described above also are ACPC synthase polynucleotides. Typically, homologous ACPC synthase poly- nucleotide sequences can be identified by hybridization of candidate polynucleotides to known ACPC synthase polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions~2X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X SSC, 0.1% SDS, 50°C once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each—homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5- 15% basepair mismatches.

Species homologs of the ACPC synthase polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of ACPC synthase polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well Icnown that the T_m of a double-stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al., J. Mol.

Biol. 81, 123 (1973). Variants of human ACPC synthase polynucleotides or ACPC synthase polynucleotides of other species can therefore be identified by hybridizing a putative homologous ACPC synthase polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO:l or 3 or the complements thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising ACPC synthase polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.

Nucleotide sequences which hybridize to ACPC synthase polynucleotides or their complements following stringent hybridization and/or wash conditions also are ACPC synthase polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.

Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20 °C below the calculated T_m of the hybrid under study. The T_m of a hybrid between an ACPC synthase polynucleotide having a nucleotide sequence shown in SEQ ID NO:l, 3, 5, 7, or 9 or the complements thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):

T_m = 81.5 °C - 16.6(log_I0[Na⁺]) + 0.41(%G + C) - 0.63(%formamide) - 600//), where / = the length of the hybrid in basepairs.

Stringent wash conditions include, for example, 4X SSC at 65°C, or 50% formamide, 4X SSC at 42°C, or 0.5X SSC, 0.1% SDS at 65°C. Highly stringent wash conditions include, for example, 0.2X SSC at 65°C.

Preparation of ACPC Synthase Polynucleotides

A naturally occurring ACPC synthase polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Poly- nucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated ACPC synthase polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprise ACPC synthase nucleotide sequences. Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.

ACPC synthase cDNA molecules can be made with standard molecular biology techniques, using ACPC synthase mRNA as a template. ACPC synthase cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of ACPC synthase polynucleotides using either human genomic DNA or cDNA as a template.

Alternatively, synthetic chemistry techniques can be used to synthesize ACPC synthase polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode an ACPC synthase polypeptide having, for example, an amino acid sequence shown in SEQ ID NO:2, 4, 6, 8, or 10 or a biologically active variant of one of those sequences.

Obtaining Full-Length ACPC Synthase Polynucleotides

The partial sequences of SEQ ID NOS:l, 3, 5, 7, or 9 or their complements can be used to identify the corresponding full length gene(s) from which they were derived.

The partial sequences can be nick-translated or end-labeled with ³²P using polynucleotide kinase using labeling methods known to those with skill in the art (BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al., eds., Elsevier Press, N.Y., 1986). For example, a lambda library prepared from human tissue can be screened directly with the labeled sequences of interest or the library can be converted en masse to pBluescript (Stratagene Cloning Systems, La Jolla, Calif. 92037) to facilitate bacterial colony screening (see Sambrook et al., 1989, pg. 1.20).

Both methods are well known in the art. Briefly, filters with bacterial colonies containing the library in pBluescript or bacterial lawns containing lambda plaques are denatured, and the DNA is fixed to the filters. The filters are hybridized with the labeled probe using hybridization conditions described by Davis et al., 1986. The partial sequences, cloned into lambda or pBluescript, can be used as positive controls to assess background binding and to adjust the hybridization and washing stringencies necessary for accurate clone identification. The resulting autoradiograms are compared to duplicate plates of colonies or plaques; each exposed spot corresponds to a positive colony or plaque. The colonies or plaques are selected and expanded, and the DNA is isolated from the colonies for further analysis and sequencing.

Positive cDNA clones are analyzed to determine the amount of additional sequence they contain using PCR with one primer from the partial sequence and the other primer from the vector. Clones with a larger vector-insert PCR product than the original partial sequence are analyzed by restriction digestion and DNA sequencing to determine whether they contain an insert of the same size or similar as the mRNA size determined from Northern blot Analysis.

Once one or more overlapping cDNA clones are identified, the complete sequence of the clones can be determined, for example after exonuclease III digestion (McCombie et al., Methods 3, 33-40, 1991). A series of deletion clones are generated, each of which is sequenced. The resulting overlapping sequences are assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a highly accurate final sequence.

Various PCR-based methods can be used to extend the nucleic acid sequences encoding the disclosed portions of human ACPC synthase to detect upstream sequences such as promoters and regulatory elements. For exampleM restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar. PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al., Nucleic Acids Res. 16, 8186, 1988). Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68°-72° C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.

Another method which can be used is capture PCR, which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al., PCR Methods Applic. 1, 111-119, 1991). In this method, multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment ofthe DNA molecule before performing PCR.

Another method which can be used to retrieve unknown sequences is that of Parker et al., Nucleic Acids Res. 19, 3055-3060, 1991). Additionally, PCR, nested primers, and PROMOTERFINDER libraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA. This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.

Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products. For example, capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity-can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.

Obtaining ACPC Synthase Polypeptides

ACPC synthase polypeptides can be obtained, for example, by purification from human nerve, liver, prostate, or colon cells; by expression of ACPC synthase polynucleotides; or by direct chemical synthesis.

Protein Purification

ACPC synthase polypeptides can be purified, for example, from human fetal liver, fetal lung, prostate, colon, or neuroepithelial (Ntera-2) cells, or from cells transfected with polynucleotides which express ACPC polypeptides. A purified ACPC synthase polypeptide is separated from other compounds which normally associate with the ACPC synthase polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. A preparation of purified ACPC synthase polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS- polyacrylamide gel electrophoresis. Enzymatic activity of the purified preparations can be assayed, for example, as described in Example 2.

Expression of ACPC synthase Polynucleotides

To express an ACPC synthase polypeptide, an ACPC synthase polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding ACPC synthase polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y, 1989.

A variety of expression vector/host systems can be utilized to contain and express sequences encoding an ACPC synthase polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translated regions of the vector — enhancers, promoters, 5' and 3' untranslated regions ~ which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding an ACPC synthase polypeptide, vectors based on SV40 or EBV can be used with an appropriate selectable marker.

Bacterial and Yeast Expression Systems

In bacterial systems, a number of expression vectors can be selected depending upon the use intended for an ACPC synthase polypeptide. For example, when a large quantity of an ACPC synthase polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPT vector, a sequence encoding an ACPC synthase polypeptide can be ligated in frame with sequences for the amino-terminal Met and the subsequent 7 residues of μ-galactosidase so that a hybrid protein is produced. pIN vectors (Van

Heeke & Schuster, J. Biol. Chem. 264, 5503-5509, 1989 or pGEX vectors (Promega, Madison, Wis.) also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, can be used.

For reviews, see Ausubel et al. (1989) and Grant et al, Methods Enzymol. 153, 516- 544, 1987.

Plant and Insect Expression Systems

If plant expression vectors are used, the expression of sequences encoding ACPC synthase polypeptides can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987). Alternatively, plant promoters such as the small subunit of

RUBISCO or heat shock promoters can be used (Coruzzi et ah, EMBO J. 3, 1671- 1680, 1984; Broglie et al, Science 224, 838-843, 1984; Winter et al, Results Probl. Cell Differ. 17, 85-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murry, in

MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191-196, 1992).

An insect system also can be used to express an ACPC synthase polypeptide. For example, in one such system Autographa californica nuclear polyhedrosis virus

(AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. Sequences encoding ACPC synthase polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of ACPC synthase polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which ACPC synthase polypeptides can be expressed (Engelhard et al, Proc. Nat. Acad. Sci. 91, 3224- 3227, 1994).

Mammalian Expression Systems

A number of viral-based expression systems can be used to express ACPC synthase polypeptides in mammalian host cells. For example, if an adenovirus is used as an expression vector, sequences encoding ACPC synthase polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing an ACPC synthase polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659, 1984). If desired, transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid. HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).

Specific initiation signals also can be used to achieve more efficient translation of sequences encoding ACPC synthase polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding an

ACPC synthase polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al., Results Probl. Cell Differ. 20, 125-162, 1994).

Host Cells

A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed ACPC synthase polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding, and/or function. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and

WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing ofthe foreign protein.

Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express ACPC synthase polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced ACPC synthase sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I. Freshney, ed., 1986. Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11, 223-32, 1977) and adenine phosphoribosylfransferase (Lowy et al., Cell 22, 817-23, 1980) genes which can be employed in tic or aprt cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. 77, 3567-70, 1980) npt confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin et al, J. Mol. Biol. 150, 1- 14, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, 1992, supra). Additional selectable genes have been described. For example, trpB, allows cells to utilize indole in place of tryptophan; hisD, allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. 85, 8047-51, 1988). Visible markers such as anthocyanins, -glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify fransformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al., Methods Mol. Biol. 55, 121-131, 1995).

Detecting Expression of ACPC Synthase Polypeptides

Although the presence of marker gene expression suggests that an ACPC synthase polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding an ACPC synthase polypeptide is inserted within a marker gene sequence, transformed cells containing sequences which encode the ACPC synthase polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding an ACPC synthase polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of an ACPC synthase polynucleotide. Alternatively, host cells which contain an ACPC synthase polynucleotide and which express an ACPC synthase polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a polynucleotide sequence encoding an ACPC synthase polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding the ACPC synthase polypeptide. Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding the ACPC synthase polypeptide to detect fransformants which contain an ACPC synthase polynucleotide.

A variety of protocols for detecting and measuring the expression of an ACPC synthase polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on an ACPC synthase polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in Hampton et al, SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn., 1990) and Maddox et al, J. Exp. Med. 158, 1211-1216, 1983).

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding ACPC synthase polypeptides include oligolabeling. nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding an ACPC synthase polypeptide can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Expression and Purification of ACPC Synthase Polypeptides

Host cells transformed with nucleotide sequences encoding an ACPC synthase polypeptide can be cultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode ACPC synthase polypeptides can be designed to contain signal sequences which direct secretion of ACPC synthase polypeptides through a prokaryotic or eukaryotic cell membrane.

As discussed above, other constructions can be used to join a sequence encoding an

ACPC synthase polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine- tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). Inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the ACPC synthase polypeptide also can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing an ACPC synthase polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IIVIAC (immobilized metal ion affinity chromatography, as described in Porath et al., Prot. Exp. Purif. 3, 263-281, 1992), while the enterokinase cleavage site provides a means for purifying the ACPC synthase polypeptide from the fusion protein. Vectors which contain fusion proteins are disclosed in Kroll et al., DNA

Cell Biol. 72, 441-453, 1993.

Chemical Synthesis

Sequences encoding an ACPC synthase polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al., Nucl. Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225- 232, 1980). Alternatively, an ACPC synthase polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifreld, J Am. Chem. Soc. 85,

2149-2154, 1963; Roberge et al, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of ACPC synthase polypeptides can be separately synthesized and combined using chemical methods to produce a full- length molecule.

The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, NY., 1983). The composition of a synthetic ACPC synthase polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion ofthe amino acid sequence ofthe ACPC synthase polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein. Production of Altered ACPC Synthase Polypeptides

As will be understood by those of skill in the art, it may be advantageous to produce ACPC synthase polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

The nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter ACPC synthase polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.

Antibodies

Any type of antibody known in the art can be generated to bind specifically to an epitope of an ACPC synthase polypeptide. "Antibody" as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab')₂, and

Fv, which are capable of binding an epitope of an ACPC synthase polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids. An antibody which specifically binds to an epitope of an ACPC synthase polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immuno- precipitations, or other immunochemical assays known in the art. Various immunoassays can be used to identify antibodies having the desired specificity.

Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen.

Typically, an antibody which specifically binds to an ACPC synthase polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably, antibodies which specifically bind to ACPC synthase polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate an ACPC synthase polypeptide from solution.

ACPC synthase polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, an ACPC synthase polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially useful.

Monoclonal antibodies which specifically bind to an ACPC synthase polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al, Nature 256, 495-497, 1985; Kozbor et al, J. Immunol. Methods 81, 31-42, 1985; Cote et al, Proc. Natl. Acad. Sci. 80, 2026- 2030, 1983; Cole et al, Mol. Cell Biol. 62, 109-120, 1984).

In addition, techniques developed for the production of "chimeric antibodies," the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al, Proc. Natl. Acad. Sci. 81, 6851-6855, 1984; Neuberger et al, Nature 312, 604-608, 1984; Takeda et al, Nature 314, 452-454, 1985). Monoclonal and other antibodies also can be "humanized" to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions. Alternatively, humanized antibodies can be produced using recombinant methods, as described in GB2188638B. Antibodies which specifically bind to an ACPC synthase polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.

Alternatively, techniques described for the production of single chain antibodies can be adapted using methods Icnown in the art to produce single chain antibodies which specifically bind to ACPC synthase polypeptides. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. SS, 11120-23, 1991).

Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al, 1996, Ewr. J. Cancer Prev. 5, 507-11). Single-chain antibodies can be mono- or bispecific, and can be bivalent or tefravalent. Construction of tefravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, J. Biol. Chem. 269, 199-206.

A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al, 1995, Int. J. Cancer 61, 497-501; Nicholls et al, 1993, J. Immunol. Meth. 165, 81- 91).

Antibodies which specifically bind to ACPC synthase polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al, Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al, Nature 349, 293-299, 1991).

Other types of antibodies can be constructed and used therapeutically in methods of the invention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.

Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which an ACPC synthase polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration. Antisense Oligonucleotides

Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of ACPC synthase gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al,

Chem. Rev. 90, 543-583, 1990.

Modifications of ACPC synthase gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of an ACPC synthase gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred.

Similarly, inhibition can be achieved using "triple helix" base-pairing methodology.

Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al, in Huber & Carr, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco, N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of an ACPC synthase polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, A, or 5 or more stretches of contiguous nucleotides which are precisely complementary to an ACPC synthase polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent ACPC synthase nucleotides, can provide sufficient targeting specificity for ACPC synthase mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular ACPC synthase polynucleotide sequence.

Antisense oligonucleotides can be modified without affecting their ability to hybridize to an ACPC synthase polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.

Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5'- substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al, Trends Biotechnol. 10, 152-158, 1992; Uhlmann et al, Chem.

Rev. 90. 543-584, 1990; Uhlmann et al, Tetrahedron. Lett. 215, 3539-3542, 1987. Ribozymes

Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515,

1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

The coding sequence of an ACPC synthase polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from the ACPC synthase polynucleotide. Methods of designing and constructing ribozymes which can cleave RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201).

Specific ribozyme cleavage sites within an ACPC synthase RNA target can be identified by scanning the ACPC synthase target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate ACPC synthase RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. The nucleotide sequences shown in SEQ ID NOS:l and 3 and their complements provide sources of suitable hybridization region sequences. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions ofthe ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region ofthe ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, Iiposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing

DNA construct into cells in which it is desired to decrease ACPC synthase expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.

As taught in Haseloff et al., U.S. Patent 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

Screening Methods

The invention provides methods for identifying modulators, i.e., candidate or test compounds which bind to ACPC synthase polypeptides or polynucleotides and/or have a stimulatory or inhibitory effect on. for example, expression or activity of the ACPC synthase polypeptide or polynucleotide. so as to regulate NMDA receptor activity. Decreased ACPC synthase activity is useful, for example, in developmental or genetic disorders characterized by inappropriately high levels of ACPC synthase activity. Increased ACPC synthase activity may be desired, for example, in chronic neurodegenerative diseases, after ischemic injury to the brain or spinal cord, or in neurological conditions such as depression or anxiety which can be treated with ACPC.

The invention provides assays for screening test compounds which bind to or modulate the activity of an ACPC synthase polypeptide or an ACPC synthase polynucleotide. A test compound preferably binds to an ACPC synthase polypeptide or polynucleotide. More preferably, a test compound decreases an ACPC synthase activity of an ACPC synthase polypeptide or expression of an ACPC synthase polynucleotide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound. Some candidate test compounds will preferably bind to a substrate or cofactor binding site on the ACPC synthase polypeptide, such as the pyridoxal phosphate binding site.

Test Compounds

Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997. Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al, J. Med. Chem. 37, 2678, 1994; Cho et al, Science 261, 1303, 1993; Carell et al, Angew. Chem. Int. Ed. Engl.

33, 2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al, J. Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented in solution (see, e.g., Houghten, Biotechniques 13, 412-421, 1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U.S. Patent 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. U.S.A.

89, 1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990; Devlin, Science 249, 404-406, 1990); Cwirla et al, Proc. Natl. Acad. Sci. 97, 6378-6382, 1990; Felici, J. Mol. Biol. 222, 301-310, 1991; and Ladner, U.S. Patent 5,223,409).

High Throughput Screening

Test compounds can be screened for the ability to bind to ACPC synthase polypeptides or polynucleotides or to affect ACPC synthase activity or ACPC synthase gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.

Alternatively, "free format assays," or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.

Another example of a free format assay is described by Chelsky, "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadephia, Pa. (Nov. 7-10, 1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.

Yet another example is described by Salmon et al., Molecular Diversity 2, 57-63 (1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.

Another high throughput screening method is described in Beutel et al, U.S. Patent

5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly such that the assays can be performed without the test samples running together.

Binding Assays

For binding assays, the test compound is preferably a small molecule which binds to and occupies the active site of an ACPC synthase polypeptide, thereby making the active site inaccessible to substrate such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules. In binding assays, either the test compound or the ACPC synthase polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to the ACPC synthase polypeptide can then be accomplished, for example, by direct counting of radioemission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.

Alternatively, binding of a test compound to an ACPC synthase polypeptide can be determined without labeling either of the interactants. For example, a micro- physiometer can be used to detect binding of a test compound with an ACPC synthase polypeptide. A microphysiometer (e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator ofthe interaction between a test compound and an ACPC synthase polypeptide. (McConnell et al, Science 257, 1906-1912, 1992).

Determining the ability of a test compound to bind to an ACPC synthase polypeptide also can be accomplished using a technology such as real-time Bimolecular

Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo et al, Curr. Opin. Struct. Biol. 5, 699-705, 1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

In yet another aspect of the invention, an ACPC synthase polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al. Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem. 268,

12046-12054, 1993; Bartel et al, Biotechniques 14, 920-924, 1993; Iwabuchi et al, Oncogene 8, 1693-1696, 1993; and Brent W094/10300) to identify other proteins which bind to or interact with the ACPC synthase polypeptide and modulate its activity.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. For example, in one construct a polynucleotide encoding an ACPC synthase polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein ("prey" or

"sample") can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with the ACPC synthase polypeptide.

It may be desirable to immobilize either an ACPC synthase polypeptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both ofthe interactants, as well as to accommodate automation ofthe assay. Thus, either the ACPC synthase polypeptide (or polynucleotide) or the test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the ACPC synthase polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to an ACPC synthase polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.

In one embodiment, an ACPC synthase polypeptide is a fusion protein comprising a domain that allows the ACPC synthase polypeptide to be bound to a solid support. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed ACPC synthase polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.

Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either an ACPC synthase polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated ACPC synthase polypeptides, polynucleotides, or test compounds can be prepared from biotin-

NHS(N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies which specifically bind to an ACPC synthase polypeptide, polynucleotides, or a test compound, but which do not interfere with a desired binding site, such as the active site of the ACPC synthase polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies which specifically bind to an ACPC synthase polypeptide or test compound, enzyme-linked assays which rely on detecting an ACPC synthase activity of the ACPC synthase polypeptide, and SDS gel electrophoresis under non-reducing conditions-

Screening for test compounds which bind to an ACPC synthase polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises an ACPC synthase polynucleotide or polypeptide can be used in a cell-based assay system. An ACPC synthase polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as N2A neuroblastoma cells, PC 12 pheochromocytoma cells, NCB-20 cells, P19 embryonic carcinoma cells, HCC hepatocellular carcinoma cells, or PC-3 prostate cancer cells, can be used. An intact cell is contacted with a test compound. Binding of the test compound to an ACPC synthase polypeptide or polynucleotide is determined as described above, after lysing the cell to release the ACPC synthase polypeptide-or polynucleotide-test compound complex.

ACPC Synthase Assays

Test compounds can be tested for the ability to increase or decrease an ACPC synthase activity of an ACPC synthase polypeptide. ACPC synthase activity can be measured, for example, by measuring the amount of ACPC produced per unit time

(Sato, T., Oeller, P.W., and Theologis, A. (1991) J. Biol. Chem. 266:3752; see Example 2) after contacting either a purified ACPC synthase polypeptide, a cell extract, or an intact cell with a test compound. A test compound which decreases ACPC synthase activity by at least about 10, preferably about 50. more preferably about 75, 90, or 100% is identified as a potential agent for decreasing ACPC synthase activity. A test compound which increases ACPC synthase activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for increasing ACPC synthase activity.

ACPC Synthase Gene Expression

In another embodiment, test compounds which increase or decrease ACPC synthase gene expression are identified. An ACPC synthase polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product ofthe ACPC synthase polynucleotide is determined. The level of expression of ACPC synthase mRNA or polypeptide in the presence ofthe test compound is compared to the .level of expression of ACPC synthase mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of ACPC synthase mRNA or polypeptide is greater in the presence ofthe test compound than in its absence, the test compound is identified as a stimulator or enhancer of ACPC synthase mRNA or polypeptide expression. Alternatively, when expression of ACPC synthase mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of ACPC synthase mRNA or polypeptide expression.

The level of ACPC synthase mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptides. Either qualitative or quantitative methods can be used. The presence of polypeptide products of an ACPC synthase polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into an ACPC synthase polypeptide. Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell which expresses an ACPC synthase polynucleotide can be used in a cell-based assay system. The ACPC synthase polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as N2A neuroblastoma cells, PCI 2 pheochromocytoma cells, NCB-20 cells, P19 embryonic carcinoma cells, HCC hepatocellular carcinoma cells, or PC-3 prostate cancer cells, can be used.

Pharmaceutical Compositions

The invention also provides pharmaceutical compositions which can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the invention can comprise, for example, an ACPC synthase polypeptide, ACPC synthase polynucleotide, antibodies which specifically bind to ACPC synthase activity, or mimetics, agonists, antagonists, or inhibitors of ACPC synthase activity. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.

In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to. oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxy- propylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers. Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co.,

Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.

Therapeutic Indications and Methods

1. Treatment of Stroke, Brain Trauma, and Other Ischemic Conditions.

NMDA receptor-blocking drugs are effective in animal models at limiting neuronal damage if administered within an hour of stroke or other ischemic injury to the brain (Schehr, R.S., Nature Biotechnol. 14:1549 (1996)). Most NMDA antagonists have a poor therapeutic index, however, leading to harmful or undesirable effects (e.g., psychotomimetic symptoms) at doses only slightly higher than therapeutic doses (Schehr, supra). ACPC is apparently devoid of adverse side effects in humans (Cherkofsky, S.C. (1995) J. Pharm. Sci. 84:1231) and exhibits no significant behavioral effects in rats at doses up to 2 g/kg (Skolnick, P., Marvizon, J. Jackson,

B., Monn, J., Rice, K., and Lewin A. (1989) Life Sci. 45:1647). Nevertheless, ACPC reduces neuronal damage in animal models of ischemia(Fossom, L. H., Von Lubitz, D.K.J.E., Lin, R.C.-S., and Skolnick, P. (1995) Neurol.Res. 17:265; Long, J.B., and Skolnick, P. (1994) Eur. J. Pharmacol. 261:295). Therefore, ACPC is a candidate drug for the treatment of ischemic conditions including stroke.

According to the invention, treatment of stroke and other ischemic conditions can be achieved through the stimulation of endogenous production of ACPC instead of, or as an adjunct to, the administration of exogenous ACPC. Thus, a small molecule or cofactor which stimulates ACPC synthase in human cells can be administered in order to boost ACPC production. It may be desirable to stimulate ACPC synthase activity by 10, 15, 20, 30, 40, 50, 70, 90, 100, 150, or 200 percent or more, resulting in a similar percentage increase in ACPC concentration in plasma, in cerebrospinal fluid, or in the interstitial fluid at the site of trauma or ischemic injury. An alternative to the administration of a small molecule activator is the use of polynucleotides or polypeptides which are capable of activating ACPC synthase or stimulating its expression. The administration of any drug to increase ACPC synthase activity to treat an ischemic condition such as stroke should be performed as soon as possible following the injury, preferably within one to two hours, and more preferably within minutes.

Chronic elevation of ACPC synthase activity also exerts a neuroprotective effect in cases of brain or spinal cord ischemia (Fossom et al., supra) and can be useful in a patient who is known to be at risk for such injury. ACPC can be neuroprotective even if administered one week before injury and is thought to involve alterations in the expression of certain subunits of the NMDA receptor (Fossom, L.H., Basile,

A.S., and Skolnick, P., (1995) Molec. Pharmacol. 48:981). Thus, any of the approaches outlined above to increase ACPC synthase activity can be performed over a period of days to weeks, or indefinitely, in a patient who is thought to be at risk for stroke or ischemia. Another approach to chronically increasing ACPC synthase activity is to introduce extra copies of the ACPC synthase gene, or another polynucleotide which encodes ACPC synthase activity, by means of a gene therapy vector. The vector can be designed or administered such that additional ACPC synthase is expressed either globally or at the site of injury.

Raising endogenous ACPC production within the body of a patient can have a number of advantages compared to direct administration of ACPC. First, a drug which acts upon a regulatory site of ACPC synthase to stimulate its enzymic activity will probably be effective and require lower dosage than would administration of ACPC itself. Second, by stimulating endogenous production, a long-lasting and continuous increase of ACPC concentration is more likely to be achieved, thereby avoiding highly variable ACPC concentrations and variable receptor occupancy which inevitably result from conventional administration of exogenous ACPC. Third, an appropriately targeted gene therapy vector can direct the expression of ACPC synthase or an activator of ACPC synthase at the site of injury, thereby reducing any systemic effects. 2. Treatment of Chronic Neurodegenerative Diseases.

Diseases characterized by chronic or progressive neurodegeneration, such as Parkinson's disease, Huntington' s disease, or Alzheimer's disease, can benefit from the use of ACPC as an antagonist of NMDA receptor activity. This is because nerve damage caused in these conditions results in the release of glutamate, with consequent excitotoxic stimulation of NMDA receptors (Carter, AJ. (1992) Drugs Future 17:595). The same treatments as described above for ischemic injury can also be applied to the treatment of neurodegenerative diseases and dementias. However, since these are chronic conditions, the continuous stimulation of ACPC synthase is preferred. Therefore, a drug which stimulates ACPC synthase activity can be administered throughout the duration of the disease, or, more preferably, a gene therapy approach can be used to elevate ACPC synthase activity for a period of days, weeks, or longer.

Treatment of Chronic Neurological Disorders.

Animal models of depression have demonstrated that ACPC has antidepressant effects, and the use of ACPC to treat humans for depression is being clinically evaluated (Papp, M., and Elzbieta, M. (1996) Eur. J. Pharmacol. 316:145). ACPC is about as effective as the commonly used antidepressant imipramine in models of chronic mild stress (Papp, supra). The antidepressant effect of ACPC is regarded as having a faster onset of action than conventional antidepressants (Papp, M., and Moryl, E. (1996) Eur. J. Pharmacol. 316:145). Furthermore, the antidepressant effects of ACPC, like its neuroprotective effects, are long-lived and may involve adaptive changes in the NMDA receptor (Skolnick, P., Layer, R.T., Trullas, R., Popik, P., Nowak, G., and Paul, LA. (1996) Phaπnacopsychiatry 29:23).

The same treatments as described above for ischemic injury can also be applied to the treatment of depression, epilepsy, or other neurological diseases which respond to treatment with ACPC. These are typically chronic conditions which demonstrate periodic exacerbations. Therefore, the strategy of administering an ACPC synthase activator to treat one of these diseases will preferably be individualized to meet the needs of the patient. For example, a short-term therapy, which involves administration of an agent (small molecule, polynucleotide, or polypeptide) to stimulate ACPC synthase, might be employed during an exacerbation of the disease in some patients. On the other hand, some patients might benefit from the continuous stimulation of ACPC synthase over days, weeks, or longer that can be achieved with a gene therapy approach (e.g., adminstration of a vector encoding an ACPC synthase activity).

4. Treatment of Alcohol Abuse.

Chronic ethanol consumption has been demonstrated to inhibit NMDA receptor activity (Lovinger, D.M., White, G., and Weight, F.F. (1989) J. Neurosci. 10:1372 ) and to upregulate NMDA receptors in certain areas ofthe brain (Gonzales, R.A., and

Brown, L.M. (1995) Life Sci. 56:571). ACPC, through its partial agonist effect at the glycine binding site of NMDA receptors, reduces ethanol consumption in rats without altering other appetitive behavior (Stromberg, M.F., Volpicelli, J.R., O'Brien, C.P., and Mackler, S.A. (1999) Pharmacol. Biochem. Behav. 64:585). Therefore, the use of agents that enhance ACPC synthase activity, as discussed above, can be beneficial to recovering alcoholics. For example, such therapies can help alleviate the neuronal hyperexcitability which accompanies alcohol withdrawal (Littleton, J. (1995) Addiction 90:1179).

5. Facilitation of Learning and Memory.

NMDA receptor function is known to be involved in learning and brain plasticity. Inhibitors of glutaminergic transmission have been found to impair repeated acquisition procedures in primates (Moerschbaecher, J.M., and Thompson, D.M. (1980) Pharmacol. Biochem. Behav. 13:887), delayed matching to sample paradigm in pigeons (McMillan, D.E. (1981) Neurotoxicology 2:485), and spatial learning in rodents tested with the three-panel runway or Morris water maze (Ohno, M., Yamamoto, T., and Watanabe, S. (1994) Eur. J. Pharmacol. 253:183; Watanabe, Y., Himi, T., Saito, H., and Abe, K. (1992) Brain Res. 582:58). Conversely, partial agonists of the glycine binding site of NMDA receptors have been found to improve spacial learning. For example, ACPC improved the performance of aged rats in the

Morris water maze (Popik, P., and Rygielska, Z. (1999) J. Physiol. Pharmacol. 50:139). Similarly, it is thought that ACPC can alleviate learning deficits in elderly humans (Popik, supra). Thus, any of the reagents or methods of the current invention which elevate ACPC synthase activity can be used to improve the cognitive function and memory of patients with learning impairment.

This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a polypeptide- binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

A reagent which affects ACPC synthase activity can be administered to a human cell, either in vitro or in vivo, to reduce ACPC synthase activity. The reagent preferably binds to an expression product of an ACPC synthase gene. If the expression product is a polypeptide, for example, the reagent can be an antibody or a small chemical compound. For treatment of human cells ex vivo, a reagent can be added to a preparation of stem cells which have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art. In one embodiment, the reagent is delivered using a liposome. Preferably, the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung or liver.

A liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell. Preferably, the transfection efficiency of a liposome is about 0.5 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, more preferably about 1.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶ cells, and even more preferably about 2.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶ cells. Preferably, a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.

Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol. Optionally, a liposome comprises a compound capable of targeting the liposome to a nerve cell, such as a nerve cell ligand exposed on the outer surface of the liposome.

Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods which are standard in the art (see, for example. U.S. Patent 5.705.151 ). Preferably, from about 0.1 μg to about 10 μg of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 μg to about 5 μg of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 μg of polynucleotides is combined with about 8 nmol liposomes.

In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al, J. Biol. Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59 (1990); Wu et al, J. Biol. Chem. 266, 338-42 (1991).

If the reagent is a single-chain antibody, a polynucleotide encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun," and DEAE- or calcium phosphate-mediated transfection.

Effective in vivo dosages of an antibody are in the range of about 5 μg to about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg of patient body weight. For administration of polynucleotides encoding single-chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg ofDNA.

If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides which express antisense oligo- nucleotides or ribozymes can be introduced into cells by a variety of methods, as described above. Preferably, a reagent reduces expression of an ACPC synthase gene or the activity of an ACPC synthase polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of an

ACPC synthase gene or the activity of an ACPC synthase polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to ACPC synthase-specific mRNA, quantitative RT-PCR, immunologic detection of an ACPC synthase polypeptide, or measurement of ACPC synthase activity.

In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act syner- gistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

Determination of a Therapeutically Effective Dose

Determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases ACPC synthase activity relative to

ACPC synthase activity which occurs in the absence of the therapeutically effective dose.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeutically effective in 50% of the population) and LD₅₀ (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅₀.

Pharmaceutical compositions which exhibit large therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate ofthe particular formulation.

Normal dosage amounts of any particular reagent can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for polypeptides or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

The above disclosure generally describes the present invention, and all patents and patent applications cited in this disclosure are expressly incorporated herein. A more complete understanding can be obtained by reference to the following specific examples which are provided for purposes of illustration only and are not intended to limit the scope ofthe invention.

EXAMPLE 1

Detection of an ACPC synthase activity

The polynucleotide of SEQ ID NO: 13 was inserted into pGEX vector and expressed as a fusion protein with glutathione S-transferase. The fusion protein was purified from lysed cells by adsorption by glutathion-agarose-beads followed by elution in the presence of free glutathione. The activity of the fusion protein (ACPC synthase polypeptide of SEQ ID NO: 14) is assessed according to the following procedures:

The fusion protein is incubated with 400 μM S-adenosylmethionine (AdoMet) (hydrogen sulfate salt) in 100 mM phosphate buffer, pH 8,0 at 30°C for 5 min; the total volume is 100 μl. The reaction is terminated by adding 500 μl of ice-cold 20 mM HgCl₂. The tubes (size 13x100 mm) are then sealed with a serum cap and the amount of aminocyclopropane-1 -carboxy late (ACC) present is determined by its conversion to ethylene. This is accomplished by injecting 200 μl of an ice-cold mixture of 5 M NaOCl an 15 M NaOH (2:1, v/v) into the tube, vortexing for 10s, then incubating the sample for 3 min and assaying for ethylene in the headspace by gas chromatography (15).

A unit of ACC synthase activity is defined as the amount ofthe fusion protein which catalyzes the formation of 1 nmol of ACC per hour under the stated conditions ofthe assay, and the specific activity is expressed as units per milligram protein. The ACPC synthase activity of the polypeptide with the amino acid sequence of SEQ ID NO: 14 is shown. EXAMPLE 2

Identification of a test compound which binds to an ACPC synthase polypeptide

Purified ACPC synthase polypeptides comprising a glutathione-S-transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution. ACPC synthase polypeptides comprise an amino acid sequence shown in SEQ ID NO:2, 4, 6, 8,10, 12, 14 or 16. The test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour.

Control samples are incubated in the absence of a test compound.

The buffer solution containing the test compounds is washed from the wells. Binding of a test compound to an ACPC synthase polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound which increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound was not incubated is identified as a compound which binds to an ACPC synthase polypeptide.

EXAMPLE 3

Identification of a test compound which increases ACPC synthase activity

A cytosolic extract from a human neuronal cell line expressing ACPC synthase is contacted with test compounds from a small molecule library and assayed for ACPC synthase activity. A control extract is also assayed in the absence of any test compound. ACPC activity is measured as the amount of ACPC produced in 30 minutes at 30°C (Sato, T., Oeller, P.W., and Theologis, A. (1991) J. Biol. Chem. 266:3752). An appropriate aliquot of the cytosolic extract is diluted into a 200 mM HEPES buffer (pH 6.5) in a 12 x 75 mm tube. The buffer also contains 40 μg of bovine serum albumin (BSA), 200 μM S-adenosylmethionine, and 10 μM pyridoxal phosphate in a total volume of 600 μl. Following the 30 minute reaction period, ACPC is quantified by the method of Lisada and Yang (Anal. Biochem. 100:140 (1979)). The ACPC synthase activity is expressed as the amount of ACPC produced (in nanomoles) per hour and per mg of extract protein. The protein concentration of the extract is determined by the method of Bradford (Anal. Biochem. 72:248 (1976)).

A test compound which increases ACPC synthase activity of the extract relative to the control extract by at least 20% is identified as an ACPC synthase activator.

EXAMPLE 4

Identification of a test compound which increases ACPC synthase gene expression

A test compound is administered to a culture of a human neuronal cell line expressing ACPC synthase and incubated at 37 °C for 10 to 45 minutes. A culture of the same type of cells incubated for the same time without the test compound provides a negative control.

RNA is isolated from the two cultures (Chirgwin et al. (1979), Biochemistry 18:5294). Northern blots are prepared using 20 to 30 _g total RNA and hybridized with a ³²P-labeled ACPC synthase-specific probe at 65° C in Express-hyb

(CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from SEQ ID NO:l. A test compound which increases the ACPC synthase-specific signal relative to the signal obtained in the absence ofthe test compound is identified as an activator of ACPC synthase gene expression.

EXAMPLE 5

Activation of ACPC synthase to enhance learning ability

A selected reagent (e.g., a test compound or a polynucleotide or polypeptide of the instant invention) is administered to rats to determine its ability to enhance spatial learning through the stimulation of ACPC synthase activity, which is evaluated using the Morris water maze (Popik, P., and Rygielska, Z. (1999) J. Physiol. Pharmacol. 50:139).

Rats are trained to find a metal platform submerged 1 cm below the surface of water in a swimming pool. The platform is positioned halfway between the wall and the center of the pool and left in this position 12 days of training. Rats are placed into the water facing the wall and allowed to find the platform. The period of time required to find the platform is measured. If desired, the spatial learning ability of a group of rats can be impaired using electroconvulsive shock (150 mA for 0.5 sec).

The selected reagent is determined to have enhanced spatial learning if it reduces the average time required for a rat to find the platform compared to a normal control (or compared to rats treated with electroconvulsive shock). The effect of the reagent on ACPC synthase activity can be estimated by comparing the improvement in maze performance using rats treated with electroconvulsive shock with the improvement obtained using ACPC (100 mg/kg i.p.).

EXAMPLE 6

Activation of ACPC synthase to treat mild chronic stress

A selected reagent (e.g., a test compound or a polynucleotide or polypeptide of the invention) is administered to rats to determine its antidepressant action through the stimulation of ACPC synthase activity. Antidepressant effect is determined using a sucrose consumption test (Papp, M., and Moryl, E. (1996) Eur. J. Pharmacol.

316:145), in which chronic mild stress decreases consumption of a 1% sucrose solution.

Rats are subjected to 12-14 hour stress periods of food or water deprivation. 45° cage tilt, intermittent illumination, soiled cage (water in sawdust bedding), paired housing, and low intensity stroboscopic illumination. The rats are first trained to consume the 1% sucrose solution, which is provided in the home cage following 14 hours of stress. Sucrose intake is calculated as the weight of the sucrose solution consumed during the test. In a positive control, a group of rats is given daily intraperitoneal injections of ACPC (100 mg/kg). In a negative control, another group of rats is injected with vehicle. Another group of rats is not stressed. The reagent is regarded as activating ACPC synthase activity if it produces a significant (at least about 20% of ACPC control) increase in consumption of the sucrose solution in the stressed animals but not in the unstressed animals.

EXAMPLE 7

Activation of ACPC synthase to treat stroke in humans

An ACPC synthase polypeptide is administered to a stroke victim within one hour of the onset of symptoms. The dose of the ACPC synthase polypeptide is selected to provide a plasma concentration of the polypeptide which approximates a concentration found to increase ACPC levels. The polypeptide elevates the concentration of ACPC in the brain tissue affected by anoxia and ischemic injury.

The elevated ACPC levels produce a partial agonist effect at the NMDA receptor which reduces neuronal death in the affected tissue. The polypeptide is administered for a period of several weeks or until the patient show signs of significant recovery of the neuronal function which was lost during the stroke.

Claims

1. An isolated polynucleotide encoding a ACPC synthase polypeptide and being selected from the group consisting of:

a) a polynucleotide encoding a ACPC synthase polypeptide comprising an amino acid sequence selected from the group consisting of:

amino acid sequences which are at least about 50% identical to

the amino acid sequence shown in SEQ ID NO: 2;

amino acid sequences which are at least about 50% identical to

the amino acid sequence shown in SEQ ID NO: 4;

the amino acid sequence shown in SEQ ID NO:4;

amino acid sequences which are at least about 50% identical to

the amino acid sequence shown in SEQ ID NO: 6;

the amino acid sequence shown in SEQ ID NO: 8; amino acid sequences which are at least about 50% identical to

the amino acid sequence shown in SEQ ID NO: 10;

5 the amino acid sequence shown in SEQ ID NO: 10;

amino acid sequences which are at least about 50% identical to

the amino acid sequence shown in SEQ ID NO: 12;

10 the amino acid sequence shown in SEQ ID NO: 12;

amino acid sequences which are at least about 50% identical to

15 the amino acid sequence shown in SEQ ID NO: 14;

the amino acid sequence shown in SEQ ID NO: 14;

amino acid sequences which are at least about 50% identical to 20 the amino acid sequence shown in SEQ ID NO: 16;

the amino acid sequence shown in SEQ ID NO: 16;

25 amino acid sequences which are at least about 50% identical to

the amino acid sequence shown in SEQ ID NO: 17; and

the amino acid sequence shown in SEQ ID NO: 17. 30 b) a polynucleotide comprising the sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 or 15;

c) a polynucleotide which hybridizes under stringent conditions to a

polynucleotide specified in (a) and (b);

e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d).

2. An expression vector containing any polynucleotide of claim 1.

3. A host cell containing the expression vector of claim 2.

4. A substantially purified ACPC synthase polypeptide encoded by a poly- nucleotide of claim 1.

5. A method for producing a ACPC synthase polypeptide, wherein the method comprises the following steps:

a) culturing the host cell of claim 3 under conditions suitable for the expression of the ACPC synthase polypeptide; and

b) recovering the ACPC synthase polypeptide from the host cell culture.

6. A method for detection of a polynucleotide encoding an ACPC synthase polypetide in a biological sample comprising the following steps: a) hybridizing any polynucleotide of claim 1 to a nucleic acid material of a biological sample, thereby forming a hybridization complex; and

b) detecting said hybridization complex.

7. The method of claim 6, wherein before hybridization, the nucleic acid material ofthe biological sample is amplified.

8. A method for the detection of a polynucleotide of claim 1 or an ACPC synthase polypeptide of claim 5 comprising the steps of:

contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the ACPC synthase polypeptide.

9. A diagnostic kit for conducting the method of any one of claims 6 to 8.

10. A method of screening for agents which decrease the activity of an ACPC synthase, comprising the steps of:

contacting a test compound with any ACPC synthase polypeptide encoded by any polynucleotide of claim 1 ;

detecting binding of the test compound of the ACPC synthase polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of a ACPC synthase.

11. A method of screening for agents which regulate the activity of an ACPC synthase, comprising the steps of: contacting a test compound with an ACPC synthase polypeptide encoded by any polynucleotide of claim 1; and

detecting an ACPC synthase activity of the polypeptide, wherein a test compound which increases the ACPC synthase activity is identified as a potential therapeutic agent for increasing the activity of the ACPC synthase, and wherein a test compound which decreases the ACPC synthase activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity ofthe ACPC synthase.

12. A method of screening for agents which decrease the activity of an ACPC synthase, comprising the steps of:

contacting a test compound with any polynucleotide of claim 1 and

detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of an ACPC synthase.

13. A method of reducing the activity of an ACPC synthase, comprising the steps of:

contacting a cell with a reagent which specifically binds to any polynucleotide of claim 1 or any ACPC synthase polypeptide of claim 4, whereby the activity of an ACPC synthase is reduced.

14. A reagent that modulates the activity of an ACPC synthase polypeptide or a polynucleotide wherein said reagent is identified by the method of any of the claims 10 to 12.

15. A pharmaceutical composition, comprising: the expression vector of claim 2 or the reagent of claim 14 and a pharmaceutically acceptable carrier.

16. Use of the pharmaceutical composition of claim 15 for modulating the activity of an ACPC synthase in a disease.

17. Use of claim 16 wherein the disease is a stroke, a nerve damage or a neurodegenerative disease.