BACKGROUND OF THE INVENTION [003] Obesity is defined as an excess of body fat relative to lean body mass. An increase in caloric intake or a decrease in energy expenditure or both can bring about this imbalance leading to surplus energy being stored as fat. Obesity is associated with important medical morbidities and an increase in mortality. The causes of obesity are poorly understood and may be due to genetic factors, environmental factors, or a combination of the two to cause a positive energy balance. In contrast, anorexia and cachexia are characterized by an imbalance in energy intake versus energy expenditure leading to a negative energy balance and weight loss. Agents that either increase energy expenditure and/or decrease energy intake, absorption, or storage would be useful for treating obesity, overweight, and associated comorbidities. Agents that either increase energy intake and/or decrease energy expenditure or increase the amount of lean tissue would be useful for treating cachexia, anorexia and wasting disorders. There is a need to identify molecules which can be regulated to treat obesity. Identification of specific genes involved in obesity will aid in the discovery of obesity targets and drugs.

[004] Stearoyl-CoA desaturase (SCD) or delta9 desaturase is a-40 KDa membrane-associated iron-containing enzyme, comprising four transmembrane domains and three highly conserved histidine box domains that are essential for the enzyme catalytic activity. SCD is part of a multienzyme complex in the endoplasmic reticulum, together with cytochrome b5 and cytochrome b5 reductase. SCD is the only enzyme in this complex sensitive to dietary changes, hormonal imbalance, developmental processes, temperature changes, metals, alcohol, peroxisomal proliferators, and phenoli compounds. Human and rodent differ markedly in both SCD gene number and transcriptional regulation, suggesting a species-specific component of regulation and function of unsaturated fatty acid metabolism. In humans, there is one gene with two transcripts (3.9 and 5.2 Kb) generated by two alternative polyadenyation sites that are expressed mainly in liver, brain, lung, and heart (Zhang et al. , Biochem. J. 340: 255-64,1999) ; while in rodent, two genes, SCD1 and SCD2, have been identified. Although SCD1 and SCD2 are highly homologous at the nucleotide and amino acid level and encode the same functional protein, their 5'flanking regions differ resulting in divergent tissue-specific gene expression. SCD1 and SCD2 are expressed in most organs with the exception of liver and brain. The SCD1 isoform is mainly in the liver and SCD2 is in the brain. SCD can be activated by differentiation of 3T3-L1 preadiocytes, up-regulated by saturated fatty acids, insulin, agouti, and ADDl/SREBPs, and down-regulated by PUPA, PPARy (Kim and Ntambi, Biochem. Biophys. Res. Comm. 266: 1-4,1999).

[005] SCD is the rate-limiting and predominant enzyme in liver, mammary gland, brain, testis, and adipose tissue in the cellular synthesis of monounsaturated fatty acids (oleate and palmitoleate) from saturated fatty acids. SCD is a membrane-associated enzyme that catalyzes the NADH-and 02-dependent desaturation of palmitate (16: 0) and stearate (18: 0) at carbon 9 to produce palmitoleate (16: 1) and oleate (18: 1), repectively. Oleate and palmitoleate are the major storage fatty acids in human adipose tissue. Oleic acid can increase VLDL secretion and accelerate atherosclerosis.

[006] It has been found that obesity is associated with increased SCD activity in liver, adipose, and skeletal muscle in various obese models (chicken, mouse, rat, and human) (Jones et al., Am. J.

Physiol. 271: E44-49,1996). SCD deficient mice demonstrate adipose tissue atrophy, and decreased levels of liver cholesterol esters and triglycerides (Miyazaki et al. , J. Biol. Chem.

275: 30132-138,2000). It is hypothesized that inhibition of SCD activity might reduce the level of triglycerides or the fat storage. In addition, inhibition of SCD activity could change the ratio of saturated to unsaturated fatty acids in cellular membranes, which can lead to changes in downstream signaling. Consistent with this hypothesis, it is known that the ratio of stearic acid to oleic acid is one of the factors influencing cell membrane fluidity, signal transduction, and cell-cell interactions. It has been shown that alteration of this ratio is important in various diseases such as aging, cancer, diabetes, obesity, hypertension, and neurological, vascular, and heart disease (Ntambi, Prog. Lipid Res. 34: 139-50,1995).

[007] The hypothalamus is the feeding-control center in mammalian brain. Many obesity-related genes have been identified which are selectively expressed in hypothalamus. To search for obesity-related novel target genes, a PCR-based differential cDNA library subtraction was established to carry out a systematic analysis of the mRNAs whose expressions were up-regulated in the hypothalamus in dietary-induced obese versus lean rats. Several genes have been identified including SCD (Figure 1). Thus, SCD is a potential obesity target for the development of agents that would be beneficial for the treatment of obesity and obesity-related disorders.

SUMMARY OF THE INVENTION [008] It is an object of the invention to provide reagents and methods for treating obesity. This and other objects of the invention are provided by one or more of the embodiments described below. One embodiment of the invention is a method of identifying potential anti-obesity agents.

For example, stearoyl-CoA desaturase (SCD) is contacted with a test compound. The test compound is identified as a potential anti-obesity agent if it alters the expression or enzymatic activity of SCD.

[009] In another embodiment of the present invention, analysis of SCD activity and test compounds are performed utilizing a 96-well VM filtration and assay plate system.

[010] Another embodiment of the invention is a method of identifying potential anti-obesity agents. A polynucleotide encoding SCD is contacted with a test compound under conditions which permit expression of SCD. The test compound is identified as a potential anti-obesity agent if it reduces transcription of SCD relative to expression of SCD in the absence of the test compound.

[011] Another embodiment of the invention is a pharmaceutical composition for treating obesity comprising a reagent that specifically binds to SCD and a pharmaceutically acceptable carrier.

[012] Yet another embodiment of the invention is a pharmaceutical composition for treating obesity comprising an antibody that specifically binds to SCD and a pharmaceutically acceptable carrier. Symptoms of the patient's obesity are thereby decreased.

[013] A further embodiment of the invention is a pharmaceutical composition for treating obesity comprising an antisense oligonucleotide that hybridizes to a polynucleotide encoding SCD and reduces expression of the polynucleotide and a pharmaceutically acceptable carrier. Even another embodiment of the invention is a method of treating obesity. An effective amount of a reagent that decreases SCD activity is administered to a patient in need thereof. Symptoms of the patient's obesity are thereby decreased.

[014] Yet another embodiment of the invention is a method of treating obesity. An effective amount of an oligonucleotide that hybridizes to a polynucleotide encoding SCD and reduces expression of the polynucleotide is administered to a patient in need thereof. Symptoms of the patient's obesity are thereby decreased.

BRIEF DESCRIPTION OF THE DRAWING [015] Figure 1. Hybridization of radioloabeled hypothalmic cDNA from diet-induced obese (DIO) and chow-fed (control) rats to target genes. Target genes are: (A) ß-actin, (B) glyceraldehyde-3-phosphate dehydrogenase (G3PDH), (C) SOCS3 (a positive control), (D) clone 1, (E) clone 2, (F) steroyl-CoA desaturase (SCD), (G) clone 3, and (H) clone 4. The expression of SOCS3, SCD, clone 2,3, and 4 transcripts is up-regulated in the obese rat hypothalamus.

[016] Figure 2. Western blot showing increased SCD level in liver microsomes prepared from rats on EFAD diet. Liver microsomes were isolated from rats fed with EFAD or chow (normal) diet. Microsomal proteins (10 pug) were resuspended in SDS lysis buffer, fractionated by SDS/PAGE, and transferred to a nitrocellulose membrane. The proteins were detected by using a polyclonal antibody against the C-terminus of rat SCD and visualized with HRP-conjugated secondary antibody.

[017] Figure 3. SCD filtration assay. A: total CPM, representing the mean + SD of 6 replicates; B: specific CPM (represents SCD enzyme activity, total CPM minus non-specific CPM in"no microsomes or-M"blank control). Abbreviations: HI, heat inactivated microsomes incubated at <BR> <BR> 100°C for 10 min ; SA, sterculic acid (100 pM) ; "NORL"-normal microsomes;"EFAD"-EFAD microsomes.

[018] Figure 4. SCD assay kinetics of EFAD rat liver microsomal protein. A: Time dependence of [3H] H20 production. Multiple sets of assays were set up in the microtiter plates and each set was terminated at indicated time. B: Dependence of [3H] H20 formation from [3H] Stearoyl-CoA on microsomal protein concentration. Aliquots of supernatants after depletion of fatty acyl-CoA by charcoal were counted for tritium. Data shown is mean + SD (n = 6).

[019] Figure 5. Dose-dependent inhibition of [3H] H20 formation from [9,10 (n)-3H] Stearoyl-CoA by SA using 5 Itg EFAD microsomal preps. A: total CPM; B: specific CPM (total CPM minus non-specific CPM in blank control). Data shown as mean + SD of 6 replicates.

[020] Figure 6. SCD filtration assay in the 96-well format. (A) [3H] H2O production in blank wells (4/plate x 5) and EFAD wells (4/plate x 5). Calculation of Z-factor is described below.

(B) Scatter plot of a representative plate showing [3H] H2O production by EFAD microsomes. In 10 of the 84 wells, SA (100 uM) was spiked prior to assay. These wells are the cluster of data points below the dashed line (which represents the 50% mean value of the EFAD specific assay signal).

[021] Figure 7. Data of five 96-well filtration VM plates. On each plate, blank (i. e. , no microsome) or normal rat liver microsomal protein were used as a control (n = 4). SCD inhibitor, sterculic acid (100 iM), was spiked (in a blinded manner) in 10 of the 96 wells.

DETAILED DESCRIPTION OF THE INVENTION [022] SCD is expressed in the hypothalamus and is significantly up-regulated in the hypothalamus of high-fat-diet induced obese rats compared with chow-fed rats. The hypothalamus is the feeding-control center, and therefore, the up-regulation of SCD in the obese rat hypothalamus indicates that it may play a role in controlling body weight and thus, may serve as an anti-obesity target.

Polypeptides [023] SCD polypeptides according to the invention comprise at least 6,8, 10,12, 15,20, 25,50, 75,100, 125, or more contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO: 2 (human) or 4 (rat), or biologically active variants thereof, as defined below. An SCD polypeptide of the invention therefore can be a portion of an SCD protein, a full-length SCD protein, or a fusion protein comprising all or a portion of an SCD protein.

Biologically Active Variants <BR> <BR> [024] SCD polypeptide variants that are biologically active, e. g. , retain enzymatic activity, also are SCD polypeptides. Preferably, naturally or non-naturally occurring SCD polypeptide variants have amino acid sequences which are at least about 50% identical to an amino acid sequence shown in SEQ ID NO: 2 or 4, or to a fragment thereof. Percent identity between a putative SCD polypeptide variant and an amino acid sequence of SEQ ID NO: 2 or 4 is determined using the Blasts alignment program (e. g. , Blosum62, Expect 10, standard genetic codes).

[025] 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 may be substituted, inserted, or deleted without abolishing biological or immunological activity of an SCD polypeptide may be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active SCD polypeptide can readily be determined by assaying for enzyme activity (see, e. g, Bioch.

Pharmacol. 55: 1045-1058,1998).

Fusion proteins [026] Fusion proteins are useful for generating antibodies against SCD polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins that interact with portions of an SCD 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.

[027] An SCD polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond. The first polypeptide segment comprises at least 6,8, 10,12, 15,20, 25, 50,75, 100,125, or more contiguous amino acids of SEQ ID NO: 2 or 4, or of a biologically active variant, such as those described above. The first polypeptide segment also can comprise full-length SCD protein.

[028] The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in fusion protein construction include p-galactosidase, 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 may be 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 may 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 may be engineered to contain a cleavage site located between the SCD polypeptide-encoding sequence and the heterologous protein sequence, so that the SCD polypeptide can be cleaved and purified away from the heterologous moiety.

[029] A fusion protein may be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two polypeptide 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: 1 (human) or 3 (rat) in proper reading frame with nucleotides encoding the second polypeptide 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).

Identif cation of Species Homologs [030] Species homologs of the SCD polypeptides disclosed herein may be obtained using SCD 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 the SCD polypeptide, and expressing the cDNAs as is known in the art.

Polynucleotides.

[031] An SCD polynucleotide may be single-or double-stranded and comprises a coding sequence or the complement of a coding sequence for an SCD polypeptide. The coding sequence for human SCD is shown in SEQ ID NO: 1. A coding sequence for rat SCD is shown in SEQ ID NO: 3. Degenerate nucleotide sequences encoding SCD polypeptides, as well as homologous nucleotide sequences which are at least about 50,55, 60,65, 70, preferably about 75,90, 96,98, or 99% identical to the nucleotide sequences shown in SEQ ID NOS: 1 or 3, or their complements also are SCD 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 affine 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 SCD polynucleotides that encode biologically active SCD polypeptides also are SCD polynucleotides.

Polynucleotide fragments comprising at least 8,9, 10,11, 12,15, 20, or 25 contiguous nucleotides of SEQ ID NO: 1 or 3, or its complements also are SCD polynucleotides. These fragments can be used, for example, as hybridization probes or as antisense oligonucleotides.

Identification of Polynucleotide Variants and Homologs [032] Variants and homologs of the SCD polynucleotides described above also are SCD polynucleotides. Typically, homologous SCD polynucleotide sequences may be identified by hybridization of candidate polynucleotides to SCD 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.

[033] Species homologs of the SCD polynucleotides disclosed herein may also 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 SCD polynucleotides can be identified, for example, by screening human cDNA expression libraries.

[034] It is well known that the Tm 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 SCD polynucleotides or SCD polynucleotides of other species can therefore be identified by hybridizing a putative homologous SCD polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO: 1 or 3, or the complement thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.

[035] Nucleotide sequences which hybridize to SCD polynucleotides or their complements following stringent hybridization and/or wash conditions also are SCD 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.

[036] Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20°C below the calculated Tm of the hybrid under study. The Tm of a hybrid between an SCD polynucleotide having a nucleotide sequence shown in SEQ ID NO 1 or 3, or the complement 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) : Tm= 81. 5°C-16. 6 (logo [Na+] ) + 0.41 (% G+C)-0. 63 (% formamide)-600/1), 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 Polynucleotides.

[037] An SCD polynucleotide may be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides may 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 SCD polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments, which comprise SCD nucleotide sequences. Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules. SCD cDNA molecules can be made with standard molecular biology techniques, using SCD mRNA as a template. 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 polynucleotides of the invention, using either human genomic DNA or cDNA as a template.

[038] Alternatively, synthetic chemistry techniques can be used to synthesize SCD polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode an SCD polypeptide having, for example, an amino acid sequence shown in SEQ ID NO: 2 or 4, or a biologically active variant thereof.

Extension of Polynucleotides [039] Various PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements. For example, 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.

[040] 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 65-72°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.

[041] 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 of the DNA molecule before performing PCR. Another method which <BR> <BR> may 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 (CLONTECH, Palo Alto, Calif.). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

[042] 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.

[043] 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) that 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 that might be present in limited amounts in a particular sample.

Obtaining Polypeptides [044] SCD polypeptides can be obtained, for example, by purification from mammalian cells, by expression of SCD polynucleotides, or by direct chemical synthesis.

Protein Purification.

[045] SCD polypeptides can be purified from any cell that expresses the polypeptide, including host cells that have been transfected with SCD expression constructs. A purified SCD polypeptide is separated from other compounds that normally associate with the SCD 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 SCD 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.

Expression of Polynucleotides [046] To express an SCD polynucleotide, the polynucleotide may be inserted into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding SCD 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<BR> MOLECULAR BIOLOGY, John Wiley & Sons, New York, N. Y. , 1989.

[047] A variety of expression vector/host systems can be utilized to contain and express sequences encoding an SCD polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmic 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<BR> (e. g. , cauliflower mosaic virus, CaMV ; tobacco mosaic virus, TMV); or with bacterial expression<BR> vectors (e. g. , Ti or pBR322 plasmids); or animal cell systems.

[048] 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 utilized, 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<BR> 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 SCD polypeptide, vectors based on SV40 or EBV can be used with an appropriate selectable marker.

Bacterial and Yeast Expression Systems [049] In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the SCD polypeptide. For example, when a large quantity of an SCD 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 the SCD polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent residues of p-galactosidase so that a hybrid protein is produced. pIN vectors (Van Heels and 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 elusion 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.

[050] 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, for example, Ausubel et al. , (1989) and Grant et al. , (Methods Enzymol. 153: 516-544,1987).

Plant and Insect Expression Systems [051] If plant expression vectors are used, the expression of sequences encoding SCD 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 <BR> <BR> al. , EMBO J. 3: 1671-16SO, 1984, Broglie et al., Science 924: 833-843,1984 ; Winter et al. , Results Probl. Cell Differ. 17: S5-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a <BR> <BR> number of generally available reviews (e. g. , Hobbs or Murray, in MCGRAW HILL YEARBOOK<BR> OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N. Y. , pp. 191-196,1992).

[052] An insect system also can be used to express an SCD polypeptide. For example, in one such system Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. Sequences encoding SCD 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 SCD polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect S. fi-ugiperda cells or Trichoplusia larvae in which SCD polypeptides can be expressed (Engelhard et al. , Proc. Nat.

Acad. Sci. 91: 3224-3227,1994).

Mantmalian Expression Systems [053] A number of viral-based expression systems can be used to express SCD polypeptides in mammalian host cells. For example, if an adenovirus is used as an expression vector, sequences encoding SCD polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence.

[054] Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus that is capable of expressing an SCD polypeptide in infected host cells (Logan and 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).

[055] Specific initiation signals also can be used to achieve more efficient translation of sequences encoding SCD polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding an SCD 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, e. g., Scharf et al. , Results Probl. Cell Differ. 20: 125-162, 1994).

Host Cells [056] A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed SCD 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 that have specific cellular machinery and characteristic mechanisms for post- translational activities (e. g. , CHO, HeLa, MDCK, HEK293, and WI3S), are available from the American Type Culture Collection (ATCC, Manassas, VA) and can be chosen to ensure the correct modification and processing of the foreign protein.

[057] Stable expression is preferred for long-term, high-yield production of recombinant proteins.

For example, cell lines which stably express SCD 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 SCD sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type (see, e. g. , ANIMAL CELL<BR> CULTURE, R. I. Freshney, ed. , 1986).

[058] 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 phosphoribosyltransferase (Lowy et al. , Cell 22: 817-23,1980) genes that can be employed in tk-or aprt~ GellsS respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dl fr confers resistance to methotrexate (Wigler et al. , Proc. Natl. Acad. Sci. 77: 3567-70,1980), npt confers resistance to the<BR> aminoglycosides, neomycin, and G-418 (Colbere-Garapin et al. , J. Mol. Biol. 150 : 1-14, 1981), and als andpat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman and Mulligan, Proc. Natl. Acad. Sci. USA 85: 8047-51,1988). Visible markers such as anthocyanins, p-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al. , Meth. Mol. Biol. 55: 121- 131,1995).

Detecting Expression [059] Although the presence of marker gene expression suggests that the SCD polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding an SCD polypeptide is inserted within a marker gene sequence, transformed cells containing sequences that encode an SCD 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 SCD polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the SCD polynucleotide.

[060] Alternatively, host cells which contain an SCD polynucleotide and which express an SCD 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 that 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 SCD polypeptide can be detected by DNA-DNA or DNA- RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding an SCD polypeptide.

[061] Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding an SCD polypeptide to detect transformants that contain an SCD polynucleotide.

[062] A variety of protocols for detecting and measuring the expression of an SCD 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 SCD polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in <BR> <BR> Hampton et al. , (SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St.<BR> <P>Paul, Minn. , 1990) andMaddox et al. , (J. Exp. Med. 158 : 1911-1216,1983).

[063] 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 SCD polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding an SCD polypeptide can be cloned into a vector for the production of an mRNA probe.

[064] Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes 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 Polypeptides [065] Host cells transformed with nucleotide sequences encoding an SCD polypeptide can be cultured under conditions suitable for the expression and recovery of the 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 SCD polypeptides can be designed to contain signal sequences which direct secretion of soluble SCD polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane- bound SCD polypeptide.

[066] As discussed above, other constructions can be used to join a sequence encoding an SCD 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 SCD polypeptide also can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing an SCD polypeptide and six histidine residues preceding a thioredoxin or an enterokinase cleavage site.

[067] The histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography, as described in Porath et al. , (Prot. Exp. Purif. 3: 263- 81, 1992). while the enterokinase cleavage site provides a means for purifying the SCD polypeptide from the fusion protein. Vectors that contain fusion proteins are disclosed in Kroll et al. , (DNA Cell. Biol. 19: 441-<BR> 453, 1993).<BR> <P>Chemical Sytzthesis [068] Sequences encoding an SCD polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see, e. g., Caruthers et al. , Nucl. Acids Res. Symp. Ser.<BR> <P>215-223, 1980; Horn et al. , Nucl. Acids Res. Symp Ser 215-223, 1980). Alternatively, an SCD polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, 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 431A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of SCD polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.

[069] The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e. g. , Creighton, PROTEINS: STRUCTURES AND<BR> MOLECULAR PRINCIPLES, WH Freeman and Co. , New York, N. Y. , 1983). The composition<BR> of a synthetic SCD polypeptide can be confirmed by amino acid analysis or sequencing (e. g. , the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the SCD 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 Polypeptides [070] As will be understood by those of skill in the art, it may be advantageous to produce SCD 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 that is longer than that of a transcript generated from the naturally occurring sequence.

[071] The nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter SCD 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 [072] Any type of antibody known in the art can be generated to bind specifically to an epitope of an SCD 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 SCD 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, for example, at least 15,25, or 50 amino acids.

[073] An antibody which specifically binds to an epitope of an SCD polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, 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 that specifically binds to the immunogen.

[074] Typically, an antibody which specifically binds to an SCD 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 SCD polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate an SCD polypeptide from solution. Most preferably, the antibodies are neutralizing antibodies, which inhibit the activity of SCD.

[075] Human SCD 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 SCD 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, <BR> <BR> 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 25 (bacilli Calmette-Guerin) and Cornyebacterium par-vus are especially useful.

[076] Monoclonal antibodies that specifically bind to an SCD 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 56: 495-497, 1985; Kozbor et al. , J. Immunol. Methods 81: 31-42,1985 ; Cote et al. , Proc. Natl. Acad. Sci.<BR> <P>80: 2026- 2030, 1993; Cole et al. , Mol. Cell Biol. 62: 109-120,1984).<BR> <P>[077] 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-<BR> 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 replacing entire complementarily determining regions. Alternatively, humanized antibodies can be produced using recombinant methods, as described in GB2188638B. Antibodies that specifically bind to an SCD polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U. S. Patent No. 5,565, 332.

[078] Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies that specifically bind to SCD 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. 88: 11170-23,1991).

[079] Single-chain antibodies also can be constructed using a DNA amplification method, such as <BR> <BR> PCR, using hybridoma cDNA as a template (Thirion et al. , 1996, Eur J. Cancer Prev. 5: 507-11).

Single-chain antibodies can be mono-or bispecific, and can be bivalent or tetravalent.

Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma and Morrison, (Nat. Biotechnol. 15: 159-63,1997). Construction of bivalent, bispecific single- chain antibodies is taught in Mallender and Voss, (J. Biol. Chem. 269: 199-206,1994).

[080] 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. , Intl. J. Cancer 61: 497-501,1995 ; Nicholls et al. , J. Immunol.

Meth. 165: Sol-91, 1993).

[081] Antibodies which specifically bind to SCD 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. 8: 3833-3S37, 1989; Winter et al., Nature 349: 293-299, 1991).

[082] 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.

[083] 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 SCD polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.

Antisense Oligonucleotides [084] Antisense oligonucleotides are nucleotide sequences that 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.

[085] 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 SCD gene products in the cell.

[086] 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, allylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters (see, e. g., Brown, Meth. Mol. Biol. 20: 1-8,1994 ; Sonveaux, Meth.. Mol. Biol. 96: 1-72,1994 ; Uhlmann et al. , Chem. Rev. 90: 543-583,1990.

[087] Modifications of SCD gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5', or regulatory regions of the SCD gene.

Oligonucleotides derived from the transcription initiation site, for example, 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., <BR> <BR> Gee et al. , in Huber & Carr, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura<BR> 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 complementarily is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of an SCD polynucleotide.

[088] Antisense oligonucleotides which comprise, for example, 2,3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to an SCD polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent SCD nucleotides, can provide sufficient targeting specificity for SCD mRNA.

[089] 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 SCD polynucleotide sequence.

[090] Antisense oligonucleotides can be modified without affecting their ability to hybridize to an SCD 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 cholesterol 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.

[091] 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., Tetrahedrons. Lett. 215: 3539-3542,1987.

Ribozymes.<BR> <P>[092] 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 and Stinchcomb, Trends Tenet. 1: 510-515,1996). Ribozymes can be used to <BR> <BR> inhibit gene function by cleaving an RNA sequence, as is known in the art (e. g. , U. S. Patent No. 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.

[093] The coding sequence of an SCD polynucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the SCD polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see, e. g., 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, e. g. , EP 321, 201).

[094] Specific ribozyme cleavage sites within an SCD RNA target can be identified by scanning the 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.

[095] Suitability of candidate SCD RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.

[096] Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

[097] Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-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 SCD 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. <BR> <BR> <P>[098] As taught in Haseloff et al. , (U. S. Patent No. 5,641, 673), ribozymes can be engineered so that ribozyme expression will occur in response to factors that 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.

Differentially Expressed Genes.

[099] Described herein are methods for the identification of genes whose products interact with SCD. Such genes may represent genes that are differentially expressed in disorders including, but not limited to, obesity. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the SCD gene or gene product may itself be tested for differential expression.

[100] The degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques. Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis.

Identification of Differentially Expressed Genes [101] To identify differentially expressed genes, total RNA or, preferably, mRNA is isolated from tissues of interest. For example, RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples (see, e. g. , Ausubel et al. , ed. , CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993). Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single- step RNA isolation process of Chomczynski, (U. S. Patent No. 4,843, 155).

[102] Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art.

They include, for example, differential screening (Tedder et al. , Proc. Natl. Acad. Sci. U. S. A.<BR> <P>85: 708-12,1988), subtractive hybridization (Hedrick et al., Nature 308: 149-53; Lee et al. , Proc.

Natl. Acad. Sci. U. S. A. 88: 2825,1984), and, preferably, differential display (Liang and Pardee, Science 257: 967-71,1992 ; U. S. Patent No. 5,262, 311).

[103] The differential expression information may itself suggest relevant methods for the treatment of disorders involving SCD. For example, treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding SCD. The differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the SCD gene or gene product are up-regulated or down- regulated.

Screening Methods [104] The invention provides assays for screening test compounds that bind to or modulate the activity of an SCD polypeptide or an SCD polynucleotide. A test compound preferably binds to an SCD polypeptide or polynucleotide. More preferably, a test compound decreases SCD expression by at least about 10, preferably about 50, more preferably about 75,90, or 100% relative to the absence of the test compound.

Test Compounds.

[105] 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 (Lam, Anticancer Drug Des. 12: 145,1997).

[106] Methods for the synthesis of molecular libraries are well known in the art (see, e. g. , DeWitt<BR> et al. , Proc. Natl. Acad. Sci. U. S. A. 90: 6909,1993 ; Erb et al., Proc. Natl. Acad. Sci. U. S. A.<BR> <P>91: 11422,1994 ; Zuckermann et al. , J. Med. Chem. 37: 2678,1994 ; Cho et al. , Science 261: 1303,<BR> 1993; Carell et al. , Angew. Chem. Int. Ed. Engl. 33 : 2059, 1994; Carell et al. , Angew. Chem. Int.<BR> <P>Ed. Engl. 33: 2061 ; Gallop et al. , J. Med. Chem. 37: 1233,1994).<BR> <P>[107] 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- <BR> <BR> 556,1993), bacteria or spores (U. S. Patent No. 5,223, 409), plasmids (Cull et al. , Proc. Natl. Acad.

Sci. U. S. A. 89: 1865-1869,1992), or phage (Scott and Smith, Science 249: 386-390,1990 ; Devlin, Science 249: 404-406,1990 ; Cwirla et al. , Proc. Natl. Acad. Sci. U. S. A. 97: 6378-6382,1990 ; Felici, J. Mol. Biol. 22: 301-310, 1991 ; and U. S. Patent No. 5,223, 409).

High Throughput Screenizg.

[108] Test compounds can be screened for the ability to bind to SCD polypeptides or polynucleotides or to affect SCD activity or SCD 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 ul. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.

[109] 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 <BR> <BR> 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.

[110] 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.

[111] Another example of a free format assay is described by Chelsky, ("Strategies for Screening <BR> <BR> Combinatorial Libraries: Novel and Traditional Approaches, "reported at the First Annual<BR> Conference of The Society for Biomolecular Screening in Philadelphia, 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.

[112] Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by W-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change. <BR> <BR> <P>[113] Yet another example is described by Salmon et al. , (Molecul. Diversity 2: 57-63,1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.

[114] Another high throughput screening method is described in Beutel et al., (U. S. Patent No.

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.

[115] For binding assays, the test compound is preferably a small molecule that binds to and occupies, for example, a catalytic domain of the SCD polypeptide, such that the enzymatic activity is inhibited. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules.

[116] In binding assays, either the test compound or the SCD 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 that is bound to the SCD polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.

[117] Alternatively, binding of a test compound to an SCD polypeptide can be determined without labeling either of the interactants. For example, a microphysiometer can be used to detect binding of a test compound with an SCD 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).

[118] Changes in this acidification rate can be used as an indicator of the interaction between a test compound and an SCD polypeptide (McConnell et al. , Science 257: 1906-1912,1992).

Determining the ability of a test compound to bind to an SCD polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63: 7338-2345,1991, 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.

[119] It may be desirable to immobilize either the SCD polypeptide or polynucleotide or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the SCD 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 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 polynucleotide 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 SCD polypeptide or polynucleotide can be accomplished in any vessel suitable for containing the reactants.

[120] Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, the SCD polypeptide is a fusion protein comprising a domain that allows the SCD 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 SCD 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.

[121] 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 SCD polypeptide or polynucleotide or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated SCD polypeptides or 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, Ill.) and immobilized in the wells of streptavidin- coated 96-well plates (Pierce Chemical). Alternatively, antibodies which specifically bind to an SCD polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the SCD polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.

[122] 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 the SCD polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the SCD polypeptide, and SDS gel electrophoresis under non-reducing conditions.

[123] Screening for test compounds which bind to an SCD polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises an SCD polypeptide or polynucleotide can be used in a cell-based assay system. An SCD polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to an SCD polypeptide or polynucleotide is determined as described above.

[124] In yet another aspect of the invention, an SCD polypeptide can be used as a"bait protein"in a two-hybrid assay or three-hybrid assay (see, e. g. , U. S. Patent No. 5,283, 317; Zervos et al., Cell<BR> 72: 223-232,1993 ; Madura et al. , J. Biol. Chem. 68: 12046-12054,1993 ; Bartel et al.,<BR> BioTechniques 14: 990-924,1993 ; Iwabuchi et al. , Oncogene 8: 1693-1696, 1993; and W094/10300), to identify other proteins which bind to or interact with the SCD polypeptide and modulate its activity.

[125] 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, polynucleotide encoding an SCD polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e. g. , GAL4). 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 that interacts with the SCD polypeptide.

[126] In another embodiment, one or two expression vectors encode two fusion proteins. The first fusion protein comprises a DNA binding domain and either a catalytic domain of an SCD molecule or a substrate of SCD. The second fusion protein comprises a transcriptional activating domain and either a catalytic domain of an SCD molecule or a substrate of SCD. If the first fusion protein comprises the SCD catalytic domain, the second fusion protein comprises the substrate, and vice versa. Optionally, the fusion proteins can comprise full-length SCD.

[127] Interaction of the two binding domains then reconstitutes a sequence-specific transcriptional activating factor. Expression of a reporter gene comprising a DNA sequence to which the DNA binding domain of the first fusion protein specifically binds is assayed in the presence of a test compound. If the test compound decreases expression of the reporter gene relative to expression of the reporter gene in the absence of the test compound, it is identified as a potential anti-obesity agent. This method can be carried out in a cell. Optionally, the fusion proteins and the reporter gene can be used in a cell-free system. Either the test compound or one of the fusion proteins can be bound to a solid support. Either can be detectably labeled.

Functional Assays.

[128] Test compounds can be tested for the ability to increase or decrease the catalytic activity of an SCD polypeptide. SCD catalytic activity can be assayed using any of the binding assays described above.

[129] Binding assays can be carried out after contacting either a purified SCD polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound that decreases the catalytic activity of an SCD polypeptide by at least about, preferably about 50, more preferably about 75,90, or 100% is identified as a potential therapeutic agent for decreasing SCD activity. A test compound which increases the catalytic activity of an SCD polypeptide by at least about 10, preferably about 50, more preferably about 75,90, or 100% is identified as a potential therapeutic agent for increasing SCD activity.

Gene Expression.

[130] In another embodiment, test compounds that increase or decrease SCD gene expression are identified. An SCD polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the SCD polynucleotide is determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of 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 mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the 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 the mRNA or polypeptide expression. The level of SCD mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used. The presence of polypeptide products of an SCD 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 SCD polypeptide. Such screening can be carried out either in a cell-free assay system or in an intact cell.

[131] Any cell that expresses an SCD polynucleotide can be used in a cell-based assay system.

The SCD 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 CHO or human embryonic kidney 293 cells, can be used.

Pharmaceutical Composition.

[132] The invention also provides pharmaceutical compositions that can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the invention can comprise, for example, an SCD polypeptide, SCD polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to an SCD polypeptide, or mimetics, activators, or inhibitors of an SCD polypeptide 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.

[133] In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically.

Pharmaceutically acceptable carriers typically are non-pyrogenic.

[134] 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.

[135] 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, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose ; gums including arable 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.

[136] 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.

[137] Pharmaceutical preparations that 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.

[138] 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.

[139] 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 that 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 that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[140] For topical or nasal administrations penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[141] The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[142] 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.

[143] Further details on techniques for formulation and administration can be found in the latest <BR> <BR> edition of REMINGTON'S PHARMACEUTICAL SCIENCES, (Mack Publishing Co. , Easton,<BR> Pa. , 20"'edition, 2000). 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.

[144] SCD, particularly human SCD, can be regulated to treat obesity. Obesity and overweight are defined as an excess of body fat relative to lean body mass. An increase in caloric intake or a decrease in energy expenditure or both can bring about this imbalance leading to surplus energy being stored as fat. Obesity is associated with important medical morbidities and an increase in mortality. The causes of obesity are poorly understood and may be due to genetic factors, environmental factors, or a combination of the two to cause a positive energy balance. In contrast, anorexia and cachexia are characterized by an imbalance in energy intake versus energy expenditure leading to a negative energy balance and weight loss. Agents that either increase energy expenditure and/or decrease energy intake, absorption or storage would be useful for treating obesity, overweight, and associated comorbidities.

[145] Agents that either increase energy intake and/or decrease energy expenditure or increase the amount of lean tissue would be useful for treating cachexia, anorexia, and wasting disorders. An SCD gene, translated proteins and agents which modulate the gene or portions of the gene or its products are useful for treating obesity, overweight, anorexia, cachexia, wasting disorders, appetite suppression, appetite enhancement, increases or decreases in satiety, modulation of body weight, and/or other eating disorders such as bulimia. Also the SCD gene, translated proteins and agents which modulate this gene or portions of the gene or its products are useful for treating obesity/overweight-associated comorbidities including hypertension, type 2 diabetes, coronary artery disease, hyperlipidemia, stroke, gallbladder disease, gout, osteoarthritis, sleep apnea and respiratory problems, some types of cancer including endometrial, breast, prostate, and colon cancer, thrombolic disease, polycystic ovarian syndrome, reduced fertility, complications of pregnancy, menstrual irregularities, hirsutism, stress incontinence, and depression.

[146] 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 an SCD polypeptide binding molecule) 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.

[147] A reagent which affects SCD activity can be administered to a human or animal cell, either in vitro or in vivo, to reduce SCD activity. The reagent preferably binds to an expression product of a human SCD gene. If the expression product is a protein, the reagent is preferably an antibody.

For treatment of human cells ex vivo, an antibody can be added to a preparation of stem cells that 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.

[148] 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, liver, spleen, heart brain, lymph nodes, and skin.

[149] 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 g of DNA per 16 nmole of liposome delivered to about 106 cells, more preferably about 1. 0) ig ofDNA per 16 nmole of liposome delivered to about 106 cells, and even more preferably about 2.0 [ig ofDNA 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.

[150] 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 particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome.

[151] Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods that are standard in the art (see, e. g., U. S. Patent No. 5,705, 151).

Preferably, from about 0. 1 ug to about 10 ag of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0. 5 ag to about 5 ug of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1. 0 ig ofpolynucleotides is combined with about 8 nmol liposomes.

[152] 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 and Wu, (J. Biol. Chem. 263: 621-24,1988) ; Wu et al. , (J. Biol. Chem.<BR> <P>269: 542-46,1994) ; Zenke et al., (Proc. Natl. Acad. Sci. U. S. A. 87: 3655-59,1990) ; Wu et al. , (J.

Biol. Chem. 66: 338-42,1991).

Detection of Therapeutic Effective Dose [153] The 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 SCD activity relative to the SCD activity which occurs in the absence of the therapeutically effective dose.

[154] 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.

[155] Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic efficacy and toxicity, for example, EDso (the dose therapeutically effective in 50% of the population) and LDso (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.

[156] Pharmaceutical compositions that 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 EDso 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.

[157] 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 that 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 of the particular formulation.

[158] Normal dosage amounts 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 proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. If the reagent is a single-chain antibody, polynucleotides 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, transfer in-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.

[159] Effective in vivo dosages of an antibody are in the range of about 5 llg to about 50 Rg/kg, about 50 ug to about 5 mg/kg, about 100 tug to about 500 wg/kg of patient body weight, and about 200 to about 250 Fg/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 ug to about 2 mg, about 5 ug to about 500 ug, and about 20 pg to about 100 ug of DNA.

[160] If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.

[161] Preferably, a reagent reduces expression of an SCD gene or the activity of an SCD 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 SCD gene or the activity of an SCD polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to SCD- specific mRNA, quantitative RT-PCR, immunologic detection of an SCD polypeptide, or measurement of SCD activity.

[162] 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 synergistically 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.

[163] 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.

Diagnostic Methods [164] SCD also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding SCD in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.

[165] Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.

[166] Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents.

Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e. g., Myers et al., Science 230: 1242,1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (e. g., Cotton et al. , Proc. Natl. Acad. Sci. USA 85: 4397-4401,1985). Thus, the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA. In addition to direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

[167] Altered levels of SCD also can be detected in various tissues. Assays used to detect levels of the SCD polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimununoassays, competitive binding assays, Western blot analysis, and ELISA assays.

[168] All patents and patent applications cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. 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 of the invention.

EXAMPLES [169] The structures, materials, compositions, and methods described herein are intended to be representative examples of the invention, and it will be understood that the scope of the invention is not limited by the scope of the examples. Those skilled in the art will recognize that the invention may be practiced with variations on the disclosed structures, materials, compositions and methods, and such variations are regarded as within the ambit of the invention.

Example 1 [170] Expression of recombinant SCD.

The Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of recombinant SCD polypeptides in yeast. The SCD encoding DNA sequence is derived from SEQ ID NO: 1 or 3. Before insertion into vector pPICZB, the DNA sequence is modified by well known methods in such a way that it contains at its 5'-end an initiation codon and at its 3'-end an enterokinase cleavage site, a His6 reporter tag and a termination codon. Moreover, at both termini, recognition sequences for restriction endonucleases are added and after digestion of the multiple cloning site of pPICZB with the corresponding restriction enzymes the modified DNA sequence is ligated into pPICZB. This expression vector is designed for inducible expression in Pichia pastoris, driven by a yeast promoter. The resulting pPICZ/md-His6 vector is used to transform the yeast.

[171] The yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea. The bound polypeptide is eluted with buffer, pH 3.5, and neutralized.

Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San Diego, CA) according to manufacturer's instructions. Purified SCD polypeptide is obtained.

Example 2 [172] Identification of test compounds that inhibit to SCD enzyme activity.

SCD membrane preps were added to 96-well microtiter plates in the present of 3H-labeled (at C9 and C10) saturated fatty acyl-CoA (substrate). Test compounds were added to the microtiter plates in a physiological buffer solution at pH 7.0.

[173] The samples were incubated for 5 minutes to one hour. Control samples were incubated in the absence of a test compound. 3H-labeled monounsaturated fatty acyl-CoA (product) was measured in an SPA assay (Microbeta). An increase in the 3H count indicated the test compound was an SCD inhibitor.

[174] Compounds that demonstrated inhibitory activity in the SPA assay were further characterized in a cell-based TLC or HPLC assay to test for potency of inhibition of SCD enzyme activity.

Example 3 [175] Measurement of SCD enzyme activity TLC assay.

In 48-well microtiter plates, liver microsomes (0.5 mg protein), test compound, and buffer were added to each well. The samples were incubated for 10 minutes at room temperature. Then, [14C] FA substrates (a mixture of [14C] 18: 0; [14C] 18: 3n-3; and [14C] 20: 3n-6) were added. The samples were incubated for 1 hour at 37°C on a rotating shaker. To saponify samples, 2.5 N KOH in methanol: water (4: 1) was added and samples were incubated for 4 hours at 65°C with shaking.

Then, formic acid (280 p1) was added to the samples to protonate the FFA, followed by the addition ofhexane (700 ul). The samples were thoroughly mixed. An aliquot (200 RI) was withdrawn from the hexane layer and spotted onto a pre-absorbent loading strip of 10% AgN03- TLC plate. Desaturase activity quantitated using Phospho-Imager.

Example 4 [176] Identifications of a test compound which decreases SCD gene expressions.

A test compound is administered to a culture of human cells transfected with an SCD expression construct and incubated at 37°C for 10 to 45 minutes. A culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.

[177] RNA is isolated from the two cultures as described in Chirgwin et al. , (Biochem. 18: 30 5294-99,1979). Northern blots are prepared using 20 to 30 ftg total RNA and hybridized with a 32P-labeled SCD-specific probe at 65°C in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO: 1 or 3. A test compound that decreases the SCD-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of SCD gene expression.

Example 5 [178] Differential expression of SCD in obese and lean rat.

SCD expression was measured in obese rats (obtained by putting animals on a high fat diet (45%) for 10 weeks), lean rats (resistant to the high fat diet), and control rats (maintained on a chow diet).

In one set of experiments, total RNA was isolated from obese (ob), control (c), and lean rat hypothalamus. cRNA probes were made and hybridized to rat GeneChip A (Affymetrix). Data were analyzed using Affymetrix software. Results of such an analysis are exemplified in Figure 1.

Example 6 [179] Filtration Assay for Measuring SCD ActivitY Various assay methods for SCD activity have been described. Thin layer chromatography or HPLC have been used to separate and measure the substrate and product of the SCD reaction <BR> <BR> (Obukowicz, et al. , Biochem. Pharmacol. 55: 1045-1058,1998). However, these assay formats are not HTS-compatible. Spectrophotometric detection of the reoxidation of reduced cytochrome b5 <BR> <BR> has also been used as an SCD assay (Strittmatter, et al. , Meth. Enzymol. 52: 97-101,1978), but this assay is also not HTS-compatible because of the rapidity of the oxidation of cytochrome b5.

Talamo and Bloch reported a test-tube assay for plant desaturase activity (Anal. Biochem. 29: 300- 304,1969), which measured the release of [3H] H2O during the enzyme-catalyzed generation of the monounsaturated fatty acyl-CoA product.

[180] In the present invention, an assay has been adapted to a 96-well filtration assay format that is HTS-compatible.

[181] Heterologous expression of active SCD has not been successful. However, liver microsomes from rats fed an Essential Fatty Acid Deficient (EFAD) diet are a plentiful source of SCD activity. SCD protein levels were markedly induced in the livers of rats fed an EFAD diet as compared to rats fed a normal chow diet (Figure 2).

[182] The SCD assay of the present invention is based on the production of [3H] H2O using a fatty acid substrate specifically labeled at the positions to be desaturated. After incubation of SCD- containing microsomes and substrate, the labeled fatty acyl-CoA species and microsomes are absorbed with charcoal and separated from [3H] H2O by centrifugation. The formation of [3H] H2O serves as a measure of SCD activity. Use of 96-well filtration and assay plates eliminates the need for a pipette-mediated transfer step and promotes an increase specific counts in the assay. This newly developed filtration method to measure SCD activity is rapid, reproducible, and sensitive. <BR> <BR> <P>LH,, O Production 4ssay Protocol in 96-Well Filtratioiz<BR> Plate : Preparation ofMicrosomes : Essential Fatty Acid Deficient diet (EFAD) -induced liver microsomes were prepared from Sprague-Dawley rats that were food-deprived (1 day) and refed with EFAD (2 days) to induce SCD expression using a method described by Ntambi (J. Biol.

Chem. 267: 10925-10930,1992). Normal rat liver microsomes were prepared similarly but from chow-fed rats. The microsomal buffer contains 250 mM sucrose, 150 mM KC1, 40 mM NaF, 100 mM sodium phosphate buffer pH 6. 8, 1.3 mM ATP, 1.5 mM reduced glutathione, 0.06 mM reduced coenzymeA, 0.33 mM nicotinamide, 4.9 mM MgCl2, 2 mM NADH, and 0.1% BSA (fatty acid-free).

[184] A MultiScreen 96-well filtration and assay plate (Millipore, Cat. #MAVMN0510) is sealed with Sigma Seal tape, and 38 RI microsomes (5 Rg protein) was added to each well. The microsomes were diluted in assay buffer. Either 1 il 70% DMSO or 1 ul of serially-diluted sterculic acid (SA, a potent and selective inhibitor of SCD) solutions were then added to each well.

The concentrations of SA were 100 uM, 33.3 « 10 uM, 3. 3 uM, 1 pM, 333 nM, and 100 nM The samples are incubated for 20 minutes at RT with moderate shaking (#4 setting using Titer Plate Shaker, Lab-Line Instruments, Inc.).

[185] Following incubation, 2 p. l [3H] Stearoyl-CoA (0. 2 uCi/, ul) were added to each well (0. 4 uCi total per well). The samples were incubated for 60 minutes at RT with shaking (#4 setting on plate shaker). Then, charcoal (Sigma, Cat. #C4386) (100 u. l) from a 125 mg/ml suspension in 10 mM EDTA and 100 mM sodium phosphate buffer (pH 6.8).

[186] The top of the plate is then sealed with Sigma Seal tape, and the plate is agitated vigorously 20 minutes at RT (#8 setting on plate shaker). The seal tape is removed from the plate bottom, and the plate is then positioned over a Wallac plate (Cat. #1450-514) containing 140 ul/well SuperMix scintillation fluid. The plates are spun for 20 minutes in tabletop centrifuge (3200 rpm), and then the plates are incubated for 20 minutes with shaking. The samples are then analyzed using a MicroBeta plate reader for tritium.

[187] The concentration-response for SA was fitted with sigmoidal curves generated by nonlinear regression analysis (Prism, GraphPad Software, Inc., San Diego, CA) to obtain ICso values. Data were expressed as means + SD, SD, and SD% values. The Z-factor value was calculated as follows (Zhang, et al. J. Biomol. Screen. 4: 67-73,1999) : Z-factor = 1- (3SD of sample + 3SD of control) / (mean of sample-mean of control).

[188l The background signal for the SCD filtration assay was about 1500 CPM in the reaction with no microsomal protein or with heat-inactivated microsomes (Figure 3A). The SCD activity may be depicted by specific CPM (i. e. , total CPM subtracted by blank CPM) as illustrated in Figure 3B. Addition of EFAD rat liver microsomal protein (5 gg) to the reaction produced a specific signal of about 2500 CPM. The corresponding specific signal with normal rat liver microsomes was about 500 CPM. This difference is consistent with the increased levels of SCD protein in the EFAD vs control rat liver microsomes.

[189] [3H] H2O production from [3H] Stearoyl-CoA by EFAD microsomes using the 96-well microtiter-plate format increased linearly with time (5-60 minutes) (Figure 4A). [3H] H2O production also increased as a function of the amount of microsomal protein (0. 2-5 gag) (Figure 4B).

[190] Sterculic acid (SA), a potent and selective inhibitor of SCD as compared to delta5-and delta6-desaturase, is a cyclopropenoic fatty acid that occurs naturally in oil from S. foetida seeds <BR> <BR> (Reiser, et al. , Biochem. Biophys. Res. Comm. 17: 8-11,1964 ; Raju, et al. , J. Biol. Chem. 242: 379- 84, 1967). SA inhibits [3H] H20 production in a dose-dependent manner with an ICso value of 5.9 uM (n = 3) (Figure 5). The [3H] H2O production was reduced by more than 90% in the presence of 100 uM SA.

[191] The reproducibility of the SCD filtration assay was assessed using 96-well assay plates. In each plate, wells contained either EFAD rat microsomes or no microsomes. The standard deviation (SD) or coefficient of variation (CV) and the assay dynamic range were determined and used to calculate the Z-factor (screening window coefficient that indicates assay quality <BR> <BR> assessment) (Zhang, et al. , J. Biomol. Screen 4: 67-73,1999). Figure 6A illustrates the individual assay signal of the wells containing blank or EFAD microsomes. The calculated Z-factor, 0.50, indicates that this SCD filtration assay is a suitable choice for HTS.

[192] SA was spiked into several wells and the SCD filtration assay was performed. The SD% values of the [3H] H2O signals were calculated, and the incidences of positive, false-positive, and false-negative inhibitor hit rates were scored (Figure 6B). The dashed line indicates the 50% mean value of the EFAD specific assay signal. A signal below this line (representing greater than 50% inhibition of enzyme activity) was scored as a positive hit. The wells containing SA were identified as positive hits; there were no false positives and no false negatives (Figure 6B).

[193] Figure 7 illustrates the SD% values for the 5 experiments. The SD% values for the EFAD and EFAD + SA wells were <8% for each experiment. The mean SD% for the EFAD and EFAD + SA wells in all 5 experiments were 6.6% and 5.8%, respectively.