JP2004524021A - Regulation of the SREBP pathway by targeting HisRS - Google Patents

Abstract

The human HisRS gene has been identified as a regulator of the SREBP pathway, and is therefore a therapeutic target for disorders related to the SREBP pathway. Methods are provided that include identifying modulators of HisRS and screening for agents that modulate the activity of HisRS.

Description

【Technical field】
[0001]
This application claims priority to US Provisional Application No. 60/261569, filed January 12, 2001, which is incorporated by reference in its entirety.
[Background Art]
[0002]
Background of the Invention
There is much interest in the pharmaceutical industry in understanding the mechanisms involved in cholesterol synthesis and metabolism, especially at the molecular level, resulting in the development of blood cholesterol-lowering drugs for the treatment and prevention of atherosclerosis. Have been. In addition, there is interest in understanding the molecular mechanisms that link lipid deficiency to insulin resistance. Hyperlipidemia and elevated levels of free fatty acids often develop in the same patient and are associated with several diseases, including obesity and insulin resistance, which are the main risk factors for progression to type 2 diabetes and cardiovascular disease. Correlates with "metabolic syndrome" as defined in the context. Current studies suggest that in addition to glucose levels, control of lipid levels is required to treat type 2 diabetes, heart disease, and other signs of metabolic syndrome (Santomauro AT et al., Diabetes (1999) 48: 1836-1841).
Recent progress has been made in understanding certain mechanisms involved in lipid metabolism in mammals. A key component is the sterol control element binding protein (SREBP) pathway. SREBPs are transcription factors that activate genes involved in the synthesis and transport of cholesterol and fatty acids. SREBP is a major mediator in insulin action in the liver and alterations in SREBPs expression and function have been described in obese and insulin resistant patients or animal models (Shimomura I et al., PNAS (1999) 96: 13656-61). Shimomura I et al., Journal of Biological Chemistry (1999) 274: 30028-32). SREBPs are also involved in the process of adipocyte differentiation, and particularly in adipocyte gene expression as a transcription factor that can promote adipogenesis in a major manner (reviewed by Spiegelman et al., Cell (1996) 87: 377-389). Has been). The function of SREBP is controlled by intracellular levels of sterols or polyunsaturated fatty acids (PUFAs) (Xu J. et al., J. Biol. Chem. (1999) 274: 23577-23583).
[0003]
In a high sterol or PUFA state, SREBPs are excluded from the nucleus by binding to the nuclear membrane and the endoplasmic reticulum (ER), thereby maintaining the inactivated state as a membrane-bound protein precursor. SREBP in its membrane-bound form has large N- and C-terminal segments facing the cytoplasm and short loops that protrude into the organelle lumen. The N-terminal domain is a basic helix-loop-helix-leucine zipper (bHLH-Zip) family of transcription factors and contains the "acid blob" typical of many transcription activators. (Brown and Goldstein, Cell (1997) 89: 331-340). The N-terminal acid blob is located after the basic helix-loop-helix-leucine zipper (bHLH-Zip), similar to those found in many other DNA-binding transcription factors.
Several components of the SREBP signaling pathway are known. In the low sterol state, the acid blob / bHLH-Zip domain of SREBP is rapidly translocated to the nucleus and released from the membrane after binding to specific DNA sequences to activate transcription. Two consecutive proteolytic cleavages are involved. The first protease, called site 1 protease (S1P), cleaves SREBP almost in the middle of the luminal loop (Sakai et al., J. Biol. Chem (1998) 273: 5785-5793).
After cleavage at site 1, a second protease, site 2 protease (S2P), cleaves the N-terminal fragment, releasing the mature N-terminal domain into the cell matrix, which rapidly enters the nucleus but apparently Part of the transmembrane domain remains attached to the C-terminus (Rawson et al., Molec Cell (1997) 1: 47-57). Mature, transcriptionally active SREBPs are rapidly degraded in a proteosome-dependent process. The combination of this proteolytic process and rapid turnover allows the SREBP system to quickly respond to changes in cell membrane components.
[0004]
The third component of the processing system of SREBPs is called SREBP cleavage activating protein (SCAP). SCAP is a large transmembrane protein that activates S1P in a low sterol state (Hua et al., Cell (1996) 87: 415-426). To date, the SREBP pathway has been studied primarily using mammalian cell cultures to isolate mutant cells deficient in controlling cholesterol metabolism or intracellular cholesterol transport. Thereafter, the mutant serves as a host for gene cloning by functional complementation. This triggered molecular cloning of the S1P, S2P and SCAP genes (Rawson et al., Supra; Hua et al., Supra; Goldstein et al., US Pat. Nos. 5,527,690 and 5,891,631 and PCT Application No. WO00 / 09677).
Relatively little is known about the further processing proteins of the SREBP pathway and the regulation of their activity. Proteins that regulate SREBP function may be excellent therapeutic targets for controlling the associated increased risk for dyslipidemia and cardiovascular disease.
Several SREBP pathway genes have been identified in vertebrates. The isolation of the Drosophila SREBP designated "HLH106" (GI079656) has been described (Theopold et al., Proc. Natl. Acad. Sci., USA, (1996) 93 (3): 1195-1199). C. elegans SREBP, and other Drosophila and C. elegans SREBP pathway genes are disclosed in WO00076308A1.
The ability to manipulate and screen the genomes of model organisms such as Drosophila and C. elegans has direct relevance to more complex vertebrate organisms due to significant evolutionary conservation of genes, pathways, and intracellular processes It provides a powerful tool for analyzing biochemical processes. Identifying novel functions of genes involved in specific pathways in such model organisms directly contributes to understanding the correlated pathways and how to regulate them in mammals (eg, Miklos GL and Rubin GM, Cell 1996, 86: 521-529). Drosophila and C. elegans do not suffer from human pathology, but various experimental models can mimic the pathology. Association of a pathological model with a modified expression of a Drosophila or C. elegans gene can identify an association between human diseases and human orthologs.
[0005]
In one example, genetic screening involves mutations (generally general) that cause mis-expression—generally decreased, increased or ectopic expression—of a known gene (“genetic entry point”). (Visible or selectable) phenotype in a vertebrate model organism. Additional genes are mutagenized in a random or targeted manner. When additional genetic mutations alter the original mutant phenotype, the gene is identified as a "modifier" that interacts directly or indirectly with the genetic entry point and its associated pathway. If the genetic entry point is an ortholog of a human gene associated with human pathology, such as a lipid metabolism disorder, screening can identify modified genes that are candidate targets for new treatments.
In screening using C. elegans SREBP as a genetic entry point, we found that T11G6.1 (GenBank identification number [GI] 7507690), the C. elegans ortholog of the human histidyl-tRNA synthetase (HisRS) gene (GI6996014), has SREBP function. Was found to be a modifier.
The primary function of histidyl tRNA synthetase (HisRS) is cystidine tRNA (tRNA^His)), Which is necessary for the incorporation of histidine into proteins. HisRS is a homodimeric enzyme with characteristic motifs in common with class II tRNA synthetases (including Gly, Pro, Ser and Thr tRNA synthetases). These enzymes are evolutionarily conserved in species ranging from bacteria to humans (Amaar, Y.G. and D.L. Baillie (1993) Nucl. Acids Res. 221: 4344-4347). In addition to their primary function, HisRS is one of several aaRS capable of synthesizing the intracellular signaling molecule diadenosine tetraphosphate (Freist, W. et al. (1999) Biol. Chem. 380: 623-646; Kisselev, et al. (1998) FEBS Lett. 427: 157-163). This molecule is abundant in pancreatic cells and is thought to inhibit ATP-sensitive K + (KATP) channels (Jovanovic, et al. (1997) Biochemical Pharmacology 54: 219-225). In pancreatic cells, inhibition of these channels is required for glucose-stimulated insulin release (this is the mechanism of action of sulfonylureas).
[0006]
In addition, HisRS has been implicated in nutrient sensing and regulation of translation through regulation of p70 S6 kinase. When activated, S6K phosphorylates the 40S ribosomal protein S6, leading to increased translation initiation of ribosomal mRNAs (Kawasome, et al. (1998) Proc. Natl. Acad. Sci. USA 95: 5033-5038). . Cells carrying a temperature-sensitive mutation in HisRS have been shown to decrease S6K activity when shifted to nonpermissive temperatures (Iiboshi, Y., et al. (1999) J. Biol Chem. 274: 1092- 1099). The mechanism of this feedback has not been elucidated, but the accumulation of deacylated tRNAs appears to result in a signal that inhibits S6K activity.
DISCLOSURE OF THE INVENTION
[Problems to be solved by the invention]
[0007]
Summary of the Invention
The invention uses an assay system comprising a HisRS polypeptide or nucleic acid; contacting the test agent with the assay system under conditions in which the system provides control activity in the absence of the test agent; and The difference between the activity deflected by and the activity of the control is deflected by the test agent in the assay system, which identifies the test agent as a candidate agent that modulates the SREBP pathway, lipid metabolism and / or adipogenesis Methods are provided for identifying candidate agents that modulate the SREBP pathway, lipid metabolism, and / or adipogenesis that detect activity. Candidate test agents include small molecule modulators, antibodies, and nucleic acid modulators such as antisense oligomers and PMOs, among others.
[Means for Solving the Problems]
[0008]
In one embodiment of the invention, the assay system comprises cultured cells or non-human animals expressing HisRS, wherein the assay system detects an agent-biased change in the SREBP pathway, lipid metabolism, and / or adipogenesis. .
In certain embodiments, the candidate HisRS modulator is identified in a cell-free or cell-based assay and detects a drug-biased change in an activity associated with the SREBP pathway, lipid metabolism, and / or adipogenesis. Assay systems are used to identify the SREBP pathway that modulates the activity of a candidate agent. In a preferred embodiment, the second assay detects an agent-biased change in activity associated with the SREBP pathway. A preferred second assay system is performed on cultured cells.
Further, the present invention provides a method of modulating the SREBP pathway in a mammalian cell, comprising contacting the cell with an agent that specifically binds a HisRS polypeptide or nucleic acid. In a preferred embodiment, the agent is administered to a mammal predicted to exhibit symptoms related to the SREBP pathway. Preferred agents include small molecule modulators, nucleic acid modulators, or antibodies.
BEST MODE FOR CARRYING OUT THE INVENTION
[0009]
Detailed description of the invention
We used the C. elegans model for the defective SREBP signal to identify the association between the HisRS and the SREBP pathway. We have also shown that reduced and eliminated SREBP activity in C. elegans (either by genetic mutation or RNA interference [RNAi]) results in a pale intestine phenotype due to reduced lipid accumulation in the gut. In addition, a strong loss of function of C. elegans SREBP causes a growth arrest phenotype in larvae (W000076308A1). Genetic screening was performed in C. elegans to identify modifiers (suppressors) of the pale intestine phenotype of SREBP. In particular, we used the RNAi feeding system to create a function-deficient SREBP phenotype and produced Escherichia coli that produced a double-stranded (ds) RNA corresponding to the SREBP gene that exhibited the pale intestine phenotype when the mutation was introduced. ("On SREBP RNAi-fed plates") resulted in the recovery of the more normal phenotypic C. elegans restoration. For screening, wild-type nematodes were mutagenized by EMS, enabling two generations to self-fertilize to produce homozygous nematodes for the newly introduced mutation. The resulting homozygotes were placed on SREBP RNAi feeding plates. C. elegans containing suppressor mutations were identified on SREBP RNAi-fed plates as having more intestinal pigmentation than the wild-type C. elegans pale intestine. The identity of the modified gene was revealed through genetic mapping, positional cloning, and phenotypic rescue of the mutant.
[0010]
We have identified the C. elegans SREBP phenotype suppressor, the ortholog of the human HisRS gene, T11G6.1 (GI7507690), as a member of the SREBP pathway. Thus, the HisRS genes (ie, nucleic acids and polypeptides) are attractive for the treatment of disorders related to lipid (eg, fatty acids and cholesterol) metabolism, adipogenesis, and / or other conditions associated with the SREBP signaling pathway. It is a drug target. In one example, treatment involves reducing a signal via the SREBP pathway to treat a condition associated with the metabolic syndrome.
The present invention provides methods for assessing the function of HisRS and methods for modulating (generally inhibiting or agonizing) HisRS activity, which are useful for further elucidating the SREBP pathway and developing diagnostic and therapeutic modalities for conditions associated with the SREBP pathway. Is provided in vitro and in vivo. As used herein, conditions associated with the SREBP signaling pathway include those conditions in which the SREBP pathway contributes to maintaining health, as well as those conditions whose course can be altered by regulation of the SREBP pathway.
[0011]
HisRS Nucleic acids and polypeptides
Human HisRS nucleic acid (cDNA) and protein sequences are provided in SEQ ID NOs: 1 and 2, respectively.
The term "HisRS polypeptide" refers to a full-length HisRS protein or fragment, or "functionally active", i.e., a derivative or fragment of a HisRS protein wherein one or more functional activities associated with the full-length, wild-type HisRS protein. Means those derivatives. As an example, a fragment or derivative may have antigenicity such that it can be used in immunoassays, such as immunization, production of inhibitory antibodies, etc., as discussed further below. Preferably, the functionally active HisRS fragment or derivative exhibits one or more biological activities associated with the HisRS protein, such as enzymatic activity, signal activity, ability to bind to a natural intracellular substrate. Preferred HisRS polypeptides exhibit enzymatic activity. In one embodiment, the functionally active HisRS polypeptide is a HisRS derivative that is capable of rescuing defective endogenous HisRS activity, such as in a cell-based or animal assay; It may be from a different species. When a HisRS fragment is used in an assay to identify a modulator, the fragment preferably comprises a C- or N-terminus or, in particular, a HisRS domain such as a catalytic domain, preferably at least 10, preferably at least 10, It contains 20, more preferably at least 25, and most preferably at least 50 amino acids adjacent to the HisRS protein. Functional domains can be identified using the PFAM program (Bateman A et al., 1999 Nucleic Acids Res 27: 260-262; website is pfam, wustl, edu).
[0012]
The term "HisRS nucleic acid" refers to a DNA or RNA molecule that encodes a HisRS polypeptide. Preferably, the HisRS polypeptide or nucleic acid or fragment thereof is human, but at least 70%, preferably at least 80%, preferably 85%, even more preferably 90%, and most preferably 95%, human HisRS. Orthologs or derivatives thereof having a percent sequence identity. As used herein, "percent (%) sequence identity" for a specified subject sequence, or a specified portion thereof, refers to the program WU-BLAST-2.0a19 using search parameters set to default values. Align the sequences and generate the maximum percent sequence as generated by Altschul et al., J. Mol. Biol. (1997) 215: 403-410; http://blast.wustl.edu/blast/README.html). Defined as the percentage of nucleotides or amino acids in the candidate derivative sequence that are identical to the nucleotides or amino acids in the subject sequence (or portions thereof specified), after introducing gaps if necessary to achieve identity. The HSP S and HSP S2 parameters are dynamic values and are set by the program itself, which depends on the particular sequence configuration and the specific database configuration in which the sequence of interest is searched. "% Identity value" is determined by dividing the number of identical nucleotides or amino acids that match by the sequence length for which the percent identity is reported. “Percent (%) amino acid sequence similarity” is determined by performing a calculation that is identical to that used to determine% amino acid sequence identity, but that includes conservative amino acid substitutions in addition to the calculated amino acids Is done. A conservative amino acid substitution is one in which the amino acid is replaced with another amino acid having similar characteristics such that the folding or activity of the protein is not significantly affected. Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; replaceable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; replaceable polar amino acids are glutamine and asparagine; The exchangeable basic amino acids are arginine, lysine and histidine; the exchangeable acidic amino acids are aspartic acid, glutamic acid; and the small exchangeable amino acids are alanine, serine, threonine, cysteine and glycine.
[0013]
Derivatives of the subject nucleic acid molecules The nucleic acid molecule comprises a sequence that hybridizes to the nucleic acid sequence of SEQ ID NO: 1. Hybridization stringency can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide, during hybridization and washing. Conditions that are commonly used include readily available procedures (eg, Current Protocol in Molecular Biology, Vol. 1, Chap.2.10, John Wiley & Sons, Publishers (1994); Sambrook et al., Molecular Cloning, Cold Spring Harbor ( 1989)). In one embodiment, the nucleic acid molecule of the invention comprises: 6 × 1 strength citric acid (SSC) (1 × SSC is 0.15 M NaCl, 0.015 M sodium citrate; pH 7.0), 5 × Denhard solution, 0.05% sodium pyrophosphate and Prehybridization of filters containing nucleic acids in a solution containing 100 μg / ml herring sperm DNA for 8 hours to overnight at 65 ° C .; a solution containing 6X SSC, 1X Denhardt solution, 100 μg / ml yeast tRNA and 0.05% sodium pyrophosphate Stringent hybridization conditions including: medium, 18-20 hours, hybridization at 65 ° C; and washing the filters at 65 ° C for 1 hour in a solution containing 0.2X SSC and 0.1% SDS (sodium dodecyl sulfate). Can hybridize with a nucleic acid molecule comprising the nucleotide of SEQ ID NO: 1. In another embodiment, a solution comprising: 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg / ml denatured salmon sperm DNA Medium, 6 hours, pretreatment of filter containing nucleic acid at 40 ° C .; 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% ficoll, 0.2% BSA, 100 μg Hybridization at 40 ° C. for 18-20 hours in a solution containing salmon sperm DNA and 10% (weight / volume) dextran sulfate; then 1 hour at 55 ° C. in a solution containing 2 × SSC and 0.1% SDS Moderately stringent hybridization conditions are used, including performing two washes with the same. Alternatively: in a solution containing 20% formamide, 5X SSC, 50 mM sodium phosphate (pH 7.6), 5X denhardt solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA for 8 hours to overnight at 37 ° C. Low stringency conditions including 18-20 hours of hybridization in the same buffer; and washing the filters in 1 × SSC at about 37 ° C. for 1 hour.
[0014]
In certain embodiments, the HisRS is an ortholog of human HisRS. Methods for identifying human orthologs of these genes are known in the art. Orthologs in different species usually retain the same function due to the presence of one or more protein motifs and / or three-dimensional structures. Orthologs are generally identified by sequence homology analysis, such as BLAST analysis using protein bait sequences. When the best hit sequence obtained from the results of forward BLAST searches the original query sequence in reverse BLAST (Huynen MA and Bork P, Proc Natl Acad Sci (1998) 95: 5849-5856; Huynen MA et al., Genome Research (2000) 10: 1204-1210), sequences are evaluated as potential orthologs. Programs for alignment of multiple sequences, such as CLUSTAL (Thompson JD et al., 1994, Nucleic Acids Res 22: 4673-4680), highlight conserved regions and / or residues of orthologous proteins and create a phylogenetic tree Can be used for In a phylogenetic tree representing multiple homologous sequences from various species (eg, searched through BLAST analysis), orthologous sequences from two species generally have all other sequences from these two species. It seems that the sequence is the closest on the phylogenetic tree. Structural threading or other analysis of protein folding (eg, using ProCeryon, Biosciences, Salzburg, Austria) also identifies potential orthologs. In evolution, if gene doubling occurs after speciation, a single gene in one species, such as Drosophila, may coincide with multiple genes (paralogs) in another species, for example, humans. As used herein, the term "ortholog" includes paralogs.
[0015]
HisRS Isolation, production, expression and misexpression of nucleic acids and polypeptides
HisRS nucleic acids and polypeptides are useful for identifying and testing agents that modulate the function of HisRS, and for other uses related to the involvement of HisRS in the SREBP pathway. HisRS nucleic acids are obtained using any available method. For example, techniques for isolating cDNA or genomic DNA sequences of interest by screening DNA libraries or using the polymerase chain reaction (PCR) are well known in the art.
A variety of methods are available for obtaining a HisRS polypeptide. Usually, the intended use for the polypeptide will dictate the details of the expression, production and purification methods. For example, production of polypeptides for use in screening for modulators requires methods that preserve the specific biological activity of these proteins, whereas production of polypeptides for the production of antibodies is The structural integrity of a particular epitope is required. Expression of polypeptides that are purified for screening or antibody production requires the addition of specific tags (ie, production of fusion proteins). Overexpression of HisRS polypeptides for cell-based assays used to evaluate HisRS functions, such as involvement in lipid metabolism, requires expression in eukaryotic cell lines with these cellular activities You. Techniques for protein expression, production, and purification are well known in the art; therefore, any suitable means are used (eg, Higgins SJ and Hames BD (eds) Protein Expression: A Practical Approach). , Oxford University Press Inc., New York 1999; Stanbury PF, etc., Principles of Fermentation Technology, 2^nd edition, Elsevier Science, New York, 1995; Doonan S (ed.) Protein Purification Protocols, Humana Press, New Jersey, 1996; Coligan JE, etc., Current Protocols in Protein Science (eds.), 1999, John Wiley & Sons, New York; US Pat. No. 6,165,992).
[0016]
The nucleotide sequence encoding a HisRS polypeptide can be inserted into any suitable vector for expression of the inserted protein-encoding sequence. The necessary transcription and translation signals, including the promoter / enhancer elements, may be derived from the native HisRS gene and / or its flanking regions, or may be heterologous. A variety of host-vector expression systems may be utilized, including, for example, mammalian cell lines infected with a virus (eg, vaccinia virus, adenovirus, etc.); insect cell lines infected with a virus (eg, baculovirus); yeast vectors Or a microorganism such as a bacterium transformed with a bacteriophage, plasmid, or cosmid DNA. Host cell strains that regulate, modify, and / or specifically process the expression of the gene product may be used.
A HisRS polypeptide may optionally be expressed as a fusion or chimeric product, linked via peptide bonds to a heterologous protein sequence. In some applications, the heterologous sequence encodes a transcriptional reporter gene (eg, GFP or other fluorescent protein, luciferase, β-galactosidase, etc.). Chimeric products can be prepared by linking the appropriate nucleic acid sequences encoding the desired amino acid sequences together to form an appropriate coding frame using standard methods and expressing the chimeric product. Chimeric products can also be prepared by protein synthesis techniques, such as using a peptide synthesizer (Hunkapiller et al., Nature (1984) 310: 105-111).
[0017]
HisRS polypeptides can be isolated and purified using standard methods (eg, ion exchange, affinity and gel exclusion chromatography; centrifugation; solubility differences; electrophoresis). Alternatively, native HisRS proteins can be purified from natural sources by standard methods (eg, immunoaffinity purification). Once the protein is obtained, it can be quantified and its activity measured by any suitable method, such as immunoassay, bioassay, or other measurement of physical properties such as crystallization.
The methods of the present invention may also use cells designed for altered expression (misexpression) of other genes involved in the HisRS or SREBP pathway, lipid metabolism, and / or lipogenesis. . As used herein, misexpression includes ectopic expression, overexpression, underexpression, and nonexpression (eg, by blocking gene knockout or possibly normal expression).
[0018]
Genetically modified animals
The method of the present invention provides that the non-human being genetically modified to alter the expression of HisRS and / or other genes known to be involved in adipogenesis, lipid metabolism, and / or the SREBP pathway Animals may be used. Preferably, the genetically modified animal is a mammal, especially a mouse or rat. Preferred non-mammalian species include zebrafish, C. elegans, and Drosophila. Preferably, the altered HisRS or other gene expression results in a detectable phenotype, such as a lipid profile as compared to a control animal that shows normal expression of the altered gene. Genetically modified animals can be used to further elucidate the SREBP pathway in animal models of adipogenesis, lipid metabolism, and / or pathologies associated with the SREBP pathway, and, as described below, and in vivo testing of candidate therapeutic agents. Can be used for
Preferred genetically modified animals are transgenic, at least some of their cells having unnatural nucleic acids present either as stable genomic insertions or extra chromosomal elements, which are typically mosaic. Preferred transgenic animals have a germline insertion that is stably transmitted to all cells of the offspring animal.
The non-natural nucleic acid is introduced into the host animal by any convenient method. Methods for producing transgenic non-human animals are well known in the art (for mice, Brinster et al., Proc. Nat. Acad. Sci. USA 1985, 82: 4438-42; US Pat. Nos. 4,736,866. , 4,870,009,
4,873,191, 6,127,598; Hogan, B., Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986); for homologous recombination, Capecchi, Science 1989, 244: 1288-1292. See Joyner et al., Nature 1989, 338: 153-156; for particle irradiation, see US Pat. No. 4,945,050; for Drosophila, Rubin and Spradling, Science (1982) 218: 348-. 53, US Pat. No. 4,670,388; for transgenic insects, see Berghammer AJ et al., Nature 1999, 402: 370-371; for zebrafish, Lin S. Methods Mol Biol. (2000); 136: See 375-3830; for fish, amphibians, and birds, see Houdebine and Chourrout, Experientia (1991) 47: 897-905; for rats, see Hammer et al., Cell (1990) 63: 1099-. 1112; for embryonic stem (ES) cells, see Teratocaricinomas and Embryonic Stem Cells, A Practical Approa. ch, EJ Robertson, ed., IRL Press (1987); for livestock see Pursel et al., Science (1989) 244: 1281-1288; for non-human animal clones, see Wilmut, I. (1997) Nature 385: 810-813, PCT publications WO 97/07668 and WO 97/07669; for a recombinase system for controlled expression of transgenes, see Lakso et al., PNAS (1992) 89: 6232-. See US Pat. No. 4,959,317 [for cre.loxP] and O'Gorman et al. (1991) Science 251: 1351-1355; US Pat. No. 5,654,182 [for FLP / FRT]. ).
Alterations of homozygosity or heterozygosity in the genome of the transgenic animal result in ectopic expression, overexpression (eg, multiple gene copies), underexpression and non-expression (eg, gene knockout or in some cases normal) (By blocking expression). In some applications, a "knock-out" animal typically undergoes homologous recombination such that alterations in the endogenous gene induce reduced function of the gene, preferably resulting in undetectable or negligible gene expression. It is manufactured using.
[0019]
HisRS Regulator
The present invention provides methods for identifying agents that interact and / or modulate the function of the HisRS and / or SREBP pathway. Such agents are useful in various diagnostic and therapeutic applications associated with the SREBP pathway, as well as in further analysis of the HisRS protein and its contribution to the SREBP pathway. Accordingly, the invention also provides a method for modulating the SREBP pathway, comprising the step of specifically altering HisRS activity by administering a HisRS interaction or modifier.
In a preferred embodiment, a HisRS modulator inhibits or enhances HisRS activity, possibly affecting normal function of HisRS, including transcription, protein expression, protein localization, and intracellular or extracellular activity. In a further preferred embodiment, the candidate SREBP pathway modulator specifically modulates the function of HisRS. The terms "specific modulator", "specifically modulate" and the like herein mean a modulator that binds directly to a HisRS polypeptide or nucleic acid, and preferably inhibits, enhances, and in some cases alters the function of HisRS. I do. The term also includes modulators that alter the interaction of HisRS with a binding partner or substrate (eg, by inhibiting binding and function of the HisRS binding partner or protein / binding partner complex).
Preferred HisRS modulators include HisRS interacting proteins, including small molecule chemicals, antibodies and other biotherapeutics, and nucleic acid modulators, including antisense oligomers and RNA. Modulators are formulated into therapeutic compositions, for example, as a composition with other active ingredients, such as in combination therapy, and / or a suitable carrier or excipient. Techniques for formulating and administering compounds are described in "Remington's pharmaceutical Sciences" Mack Publishing Co., Easton, PA, 19^th Found in edition.
[0020]
Small molecule regulator
Chemical agents referred to in the art as "small molecule" compounds are typically organic, non-peptidic molecules, having a molecular weight of less than 10,000, preferably less than 5,000, more preferably less than 1,000, and most preferably Has less than 500. This class of modulators includes chemically synthesized molecules, for example, compounds from combinatorial chemical libraries. Synthetic compounds are rationally designed or identified based on known or inferred characteristics of the HisRS protein, or identified by screening a compound library. Other suitable modulators of this class are natural products, especially secondary metabolites from organisms such as plants or fungi, and also identified by screening compound libraries for HisRS modulatory activity. Is done. Methods for producing and obtaining compounds are well known in the art (Schreiber SL, Science (2000) 151: 1964-1969; Radmann J and Gunther J, Science (2000) 151: 1947-1948).
Small molecule modulators identified from the screening assays described below can be used as lead compounds for which candidate clinical compounds are designed, optimized and synthesized. Such clinical compounds have utility in treating conditions associated with the SREBP pathway. The activity of candidate small molecule modulators is improved several fold through repetitive secondary functional validation, such as structure determination and candidate modulator modification and testing, as described further below. In addition, candidate clinical compounds are produced with particular attention to clinical and pharmacological characteristics. For example, reagents are derivatized and rescreened using in vitro and in vivo assays that optimize activity and minimize toxicity for drug development.
[0021]
Protein regulator
HisRS interacting proteins are endogenous, that is, they genetically or biochemically interact normally with HisRS, such as, for example, members of the HisRS pathway that regulate the expression, localization, and / or activity of HisRS. Is what you do. HisRS modulators include the dominant negative forms of the HisRS interacting protein and the HisRS protein itself. The yeast two-hybrid method and mutant screening provide a preferred method for identifying endogenous HisRS interactions (Finley, RL et al. (1996) in DNA Cloning-Expression Systems: A Practical Approach, eds. Glover D. & Hames BD (Oxford University Press, Oxford, England), pp. 169-203; Fashema SF et al., Gene (2000) 250: 1-14; Drees BL Curr Opin Chem Biol (1999) 3: 64-70; Vidal M and Legrain P Nucleic Acids Res (1999) 27: 919-29; and US Pat. No. 5,928,868). Mass spectrometry provides an alternative and preferred method for the elucidation of protein complexes (eg, Pandley A and Mann M, Nature (2000) 405: 837-846; Yates JR [3D,] Trends Genet (2000) 16: 5-8). Alternatively, the HisRS interacting protein may be a T cell receptor (Harlow and Lane, 1988, supra).
[0022]
Antibody modulator
In a preferred embodiment, the HisRS interacting protein is an antibody. Antibodies that specifically bind to a HisRS polypeptide are produced using known methods. Preferably, the antibody is specific for a mammalian HisRS polypeptide, more preferably, for human HisRS. Antibodies include polyclonal, monoclonal (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ')_TwoFragments, fragments produced by a FAb expression library, anti-idiotypic (anti-Id) antibodies, and any of the epitope binding fragments described above. Ten⁸M^-1Preferably 10⁹M^-1From 10^TenM^-1Alternatively, a monoclonal antibody having a higher affinity can be prepared as described above (Harlow E and Lane D, 1988 Antibodies: A Laboratory Manual, CSH Laboratory Press; (Harlow E and Lane D, 1999 Using Antibodies: A Laboratory Manual, CSH Laboratory Press). Laboratory Press; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York; and US Pat. Nos. 4,381,292; 4,451,570; and 4,618,577), and can be made by standard procedures. , May be made against crude cell extracts of HisRS, or substantially purified fragments thereof.If a HisRS fragment is used, preferably at least 10, more preferably at least 20 contiguous amino acids of the HisRS protein In a specific embodiment, the HisRS-specific antigen and / or immunogen is linked to a carrier protein that stimulates an immune response, eg, the polypeptide of interest is a keyhole. A limpet hemocyanin (KLH) carrier is covalently linked and the conjugate is emulsified in Freund's complete adjuvant to enhance the immune response. Immunized according to the procedure.
[0023]
Chimeric antibodies specific to HisRS polypeptides comprising different portions from different animal species can be generated. For example, the constant region of a human immunoglobulin may be linked to the variable region of a mouse mAb such that the antibody derives its biological activity from a human antibody and its binding specificity from a mouse fragment. Chimeric antibodies are produced by simultaneously splicing genes encoding appropriate regions from various sources (Morrison et al., Proc. Natl. Acad. Sci. (1984) 81: 6851-6855; Neuberger et al., Nature (1984). 312: 604-608; Takeda et al., Nature (1985) 31: 452-454). A humanized antibody, which is a form of a chimeric antibody, comprises the complementarity determining regions (CDRs) of a mouse antibody (Carlos, TM, JM Harlan. 1994. Blood 84: 2068-2101) in the background of a human framework region and a constant region. It can be produced by transplanting recombinant DNA technology (Riechmann LM, et al., 1988 Nature 323: 323-327). Since humanized antibodies contain ~ 10% mouse sequences and ~ 90% human sequences, they further reduce or eliminate immunogenicity while retaining antibody specificity (Co MS, and Queen C. 1991 Nature 351). 501-501; Morrison SL. 1992 Ann. Rev. Immun. 10: 239-265). Humanized antibodies and methods for their production are well known in the art (US PAT NO: 5,530,101; US PAT NO: 5,585,089; US PAT NO: 5,693,762, and US PAT NO: 6,180,370).
[0024]
Recombinants can produce HisRS-specific single-chain antibodies, which are single-chain polypeptides formed by linking the heavy and light chains of the Fv region via an amino acid bridge (US Pat. 4,946,778; Bird, Science (1988) 242: 423-426; Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85: 5879-5883; and Ward et al., Nature (1989) 334: 544-546. ).
Another suitable technique for antibody production involves exposing lymphocytes in vitro to a selection of an antibody library in an antigenic polypeptide or phage or similar vector (Huse et al., Science (1989) 246). : 1275-1281).
As used herein, T cell antigen receptors are included within the scope of antibody modulators (Harlow and Lane, 1988, supra).
The polypeptides and antibodies of the present invention are used with or without modification. Frequently, polypeptides and antibodies are labeled by providing a detectable signal or by covalently or non-covalently binding a substrate that is toxic to cells upon expression of the protein of interest ( Menard S, et al., Int J. Biol Markers (1989) 4: 131-134). Various labeling and conjugation techniques are known and have been extensively reported in the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent components, fluorescent lanthanide metals, chemiluminescent components, bioluminescent components, magnetic particles, and the like (US Pat.Nos. 3,817,837; 3,850,752). 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241). Alternatively, recombinant immunoglobulin may be produced (U.S. Pat. No. 4,816,567). Antibodies to cytoplasmic proteins are delivered by binding to transmembrane toxin proteins and reach their targets (U.S. Pat. NO. 6,086,900).
[0025]
When used therapeutically for a patient, the antibodies of the invention are typically administered parenterally, preferably at the target site, or intravenously. Therapeutically effective dosages and dosing regimens will be determined by clinical trial. Typically, the amount of antibody administered will range from about 0.1 mg / kg to about 10 mg / kg of the patient's body weight. For parenteral administration, the antibodies will be formulated in a unit dosage injectable form (eg, solution, suspension, emulsion) with a pharmaceutically acceptable vehicle. Such media are essentially non-toxic and non-therapeutic. Examples include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as fixed oils, ethyl oleate, or liposome carriers are also used. The vehicle contains minor amounts of additives such as buffers and preservatives, which enhance isotonicity and chemical stability, or other therapeutic capabilities. The concentration of the antibody in such a medium is typically in the range from about 1 mg / ml to about 10 mg / ml. Immunotherapy is further described in the literature (US Pat. No. 5,859,206; WO0073469).
[0026]
Nucleic acid regulator
Other preferred HisRS modulators include nucleic acid molecules that generally inhibit HisRS activity, such as antisense oligomers or double-stranded RNA (dsRNA).
Preferred antisense oligomers interfere with the function of the HisRS nucleic acid, such as DNA replication, transcription, HisRS RNA transfer, translation of protein from HisRS RNA, RNA splicing, and any catalytic activity involving HisRS RNA. In certain embodiments, the antisense oligomer is an oligonucleotide that is sufficiently complementary to bind to HisRS mRNA and preferably inhibits translation from HisRS mRNA by binding to the 5 'untranslated region. . HisRS-specific antisense oligonucleotides are preferably in the range of at least 6 to 200 nucleotides. In certain embodiments, the oligonucleotide is preferably at least 10, 15, or 20 nucleotides in length. In other embodiments, the oligonucleotide is preferably at least 50, 40, or 30 nucleotides in length. The oligonucleotide can be DNA or RNA, a chimeric mixture of DNA and RNA, derivatives or modified forms thereof, single-stranded or double-stranded. Oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone. Oligonucleotides may include other additional groups such as peptides, agents that facilitate transport across cell membranes, cleavage agents that trigger hybridization, and intercalating agents.
In another embodiment, the antisense oligomer is a phosphorothioate morpholino oligomer (PMO). PMOs are constructed from four different morpholino subunits, each containing one of the genetic bases (A, C, G, or T) linked to a six-part morpholine ring. The polymers of these subunits are linked by non-ionic phosphodiamidate intersubunit linkages. Methods for producing and using PMOs and other antisense oligonucleotides are well known in the art (eg, W099 / 18193; Summerton J, and Weller D, Antisense Nucleic Acid Drug Dev 1997, 7: 187-95). Probst JC, Methods 2000, 22: 271-281; US PAT NO: 5,325,033; US PAT NO: 5,378,841).
[0027]
Antisense oligomers are commonly used as research reagents, diagnostics, and therapeutics. For example, antisense oligonucleotides specifically inhibit gene expression and are often used to elucidate the function of particular genes (see, eg, US Pat. No. 6,165,790). Antisense oligomers are also used, for example, to distinguish between the functions of various members of a biological pathway. Antisense oligomers have been used as therapeutic ingredients in the treatment of disease states in animals and humans and have been shown to be safe and effective by many clinical trials (Milligan JF et al., 1993, J Med Chem 36: 1923). -1937; Tonkinson JL et al., 1996, Cancer Invest 14: 54-65). Thus, in one aspect of the invention, a HisRS-specific antisense oligomer is used in an assay to further elucidate the function of HisRS in the SREBP pathway. In another aspect of the invention, a HisRS-specific antisense oligomer is used as a therapeutic for the treatment of a metabolic condition.
Alternative preferred HisRS modulators are double-stranded RNA species that mediate RNA interference (RNAi). RNAi is a sequence-specific, post-transcriptional gene silencing process in animals and plants that is initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the gene to be silenced. Methods related to the use of RNAi to silence genes in C. elegans, Drosophila, plants and mammals are known in the art (Fire A, et al., 1998 Nature 391: 806-811; Fire, A. Trends Genet. 15,358-363 (1999); Sharp, PA RNA interference 2001. Genes Dev. 15,485-490 (2001); Hammond, SM, et al., Nature Rev Genet 2,110-1119 (2001); Tuschl, T. Chem. Biochem. 2,239-245 (2001); Hamilton, A. et al., Science 286,950-952 (1999); Hammond, SM, et al., Nature 404,293-296 (2000); Zamore, PD, et al., Cell 101, 25-33 ( 2000); Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, SM, et al., Genes Dev. 15,188-200 (2001); WO0129058; W09932619, and Elbashir SM, et al., 2001, Nature 411: 494. -498).
[0028]
Assay system
The present invention provides assay systems for identifying specific modulators of HisRS activity. As used herein, an "assay system" includes all components necessary to perform and analyze the results of an assay that detects and / or measures a particular phenomenon or phenomena. Generally, a first assay is used to identify or confirm the specific biochemical or molecular effects of a modulator on a HisRS nucleic acid or protein. In general, the second assay further assesses the activity of the HisRS modulator identified by the first assay, wherein the modulator affects HisRS in a manner associated with the SREBP pathway, lipid metabolism and / or lipogenesis. Make sure that In some cases, the HisRS modulator may be identified in the "first assay" in the "second assay" and tested directly without confirmation.
In a preferred embodiment, the assay system comprises contacting the candidate agent with a suitable assay system comprising a HisRS polypeptide or nucleic acid under conditions in which the system provides control activity in the absence of the agent; It is based on the specific molecular phenomenon detected by the assay system. The method further comprises detecting a similar type of activity in the presence of the candidate agent ("activity biased by the agent of the system"). The difference between the drug-biased activity and the control activity indicates that the candidate drug modulates HisRS activity and consequently the SREBP pathway. The differences used here are statistically significant. The assay system generally includes positive and / or negative controls well known in the art.
[0029]
First assay
The type of modulator tested is generally determined by the type of first assay.
The first assay for small molecule modulators
For small molecule modulators, screening assays are used to identify candidate modulators. Screening assays may be cell-based or may use cell-free systems that reproduce and retain the relevant biochemical reactions of the target protein (Sittampalam GS et al., Curr Opin Chem Biol (1997) 1: 384-91 And the accompanying literature). As used herein, the term "cell-based" refers to an assay using living cells, dead cells, or a specific subcellular fraction, such as a membrane, endoplasmic reticulum, or mitochondrial fraction. The term "cell-free" includes assays that use substantially purified proteins (either endogenous or made by recombinant methods), partially purified cell extracts, or crude extracts. . Screening assays include protein-DNA interaction, protein-protein interaction (eg, receptor-ligand binding), transcriptional activity (eg, using a reporter gene), enzymatic activity (eg, via the nature of the substrate) Detect various molecular phenomena, including changes in second messenger activity, immunogenicity and cellular morphology or other cellular properties. Suitable screening assays involve a wide range of methods, including fluorescence, radioactivity, colorimetric, spectrophotometric, and amperometric methods, to provide output on the particular molecular phenomenon to be detected. use.
In a preferred embodiment, the screening assays use fluorescence techniques, including fluorescence polarization, time-resolved fluorescence, and fluorescence resonance energy transfer. These systems provide a means to monitor protein-protein or DNA-protein interactions where the intensity of the signal emitted from the labeled dye molecule depends on the interaction with the partner molecule (e.g., Selvin PR, Nat Struct Biol. (2000) 7: 730-4; see Fernandes PB, Curr Opin Chem Biol (1998) 2: 597-603; Hertzberg RP and Pope AJ, Curr Opin Chem Biol (2000) 4: 445-451).
Suitable assay formats adapted to screen for HisRS modulators are known in the art. The primary function of HisRS is to bind histidine and histidine-tRNA. Thus, a suitable assay would detect the aminoacylation activity of this enzyme (eg, Yan W et al., 1996, Biochemistry 35: 6559-68). Preferred screening assays are high-throughput or ultra-high-throughput and thus provide an automated and cost-effective means of screening compound libraries for lead compounds (Fernandes PB, 1998, supra; Sundberg SA, Curr Opin Biotechnol 2000, 11: 47-53).
[0030]
Cell-based screening assays usually require a system for recombinant expression of HisRS and ancillary proteins required in certain assays. Cell-free assays often use purified or substantially purified proteins produced recombinantly. Suitable methods for producing recombinant proteins are of sufficient purity to retain their relevant biological activity, to be of sufficient purity to optimize activity, and to ensure reproducibility of the assay. To produce. The yeast two-hybrid method and mutant screening and mass spectrometry provide a preferred method for determining protein-protein interactions and elucidating protein complexes. In some applications, when a HisRS interacting protein is used in a screening assay, the binding specificity of the interacting protein with the HisRS protein is determined by the binding equilibrium constant (typically⁷ M^-1, Preferably at least about 10⁸ M^-1And more preferably at least about 10⁹ M^-1Are assayed by a variety of known methods, including immunogenic properties. Enzymes and receptors are assayed by substrate and ligand processing, respectively.
Screening assays measure the activity of a HisRS polypeptide, its fusion protein, or the ability of a candidate agent to specifically bind to or modulate cells or membranes carrying the polypeptide or fusion protein. HisRS polypeptides may be full length or fragments thereof that retain functional HisRS activity. HisRS polypeptides may be fused to other polypeptides, such as peptide tags for detection or anchoring, or other tags. The HisRS polypeptide is preferably human HisRS, and is an ortholog or derivative thereof as described above. In a preferred embodiment, the screening assay detects modulation of the interaction of the HisRS based on the candidate agent with a binding target, such as an endogenous or exogenous protein or other substrate having HisRS-specific binding activity, and Can be used to assess the function of various HisRS genes.
Certain screening assays are also used to test antibodies and nucleic acid modulators; for nucleic acid modulators, a suitable assay system is one that involves HisRS mRNA expression.
[0031]
The first assay for antibody modulators
For antibody modulators, a suitable first assay is a binding assay that tests the affinity and specificity of the antibody for the HisRS protein. Methods for testing antibody affinity and specificity are well known in the art (Harlow and Lane, 1988, 1999, supra). Enzyme-linked immunosorbent assay (ELISA) is a preferred method for detecting HisRS-specific antibodies; others include FACS assays, radioimmunoassays, and fluorescence assays.
The first assay for nucleic acid modulators
For nucleic acid modulators, the first assay tests the ability of the nucleic acid modulator to inhibit HisRS gene expression, preferably RNA expression. In general, expression analysis involves comparing the expression of HisRS in similar cell populations (eg, two pools that express HisRS endogenously or recombinantly) in the presence and absence of nucleic acid modulators. It is. Methods for analyzing mRNA and protein expression are well known in the art. For example, Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR (eg, TaqMan®, PE Applied Biosystems), or microarray analysis reduces HisRS mRNA expression in cells treated with a nucleic acid regulator. (Eg, Current Protocols in Molecular Biology (1994) Ausubel FM et al., Eds., John Wiley & Sons, Inc., chapter 4; Freeman WM et al., Biotechniques (1999) 26: 112- 125; Kallioniemi OP, Ann Med 2001, 33: 142-147; Blohm DH and Guiseppi-Elie, ACurr Opin Biotechnol 2001, 12: 41-47). Also, protein expression may be monitored. Proteins are most commonly detected by specific antibodies or antisera directed against either the HisRS protein or specific peptides. Various means are available, including Western blotting, ELISA, or in situ detection (Harlow E and Lane D, 1988 and 1999, supra).
[0032]
Second assay
The second assay is used to further assess the activity of the HisRS modulator identified by any of the methods described above to confirm that the modulator affects HisRS in a manner related to the SREBP pathway. . As used herein, a HisRS modulator includes a clinical candidate compound or other agent derived from a previously identified modulator. Also, a second assay may be used to test the activity of a modulator on a particular genetic or biochemical pathway, or to test the specificity of a modulator's interaction with HisRS.
The second assay generally compares similar cell or animal populations (eg, two pools of cells or animals that express endogenously or recombinantly HisRS) in the presence or absence of the candidate modulator. Usually, such assays show that treatment of cells or animals with a candidate HisRS modulator alters the SREBP pathway, lipid metabolism and / or lipogenesis as compared to untreated (or mock or placebo-treated) cells or animals. Test whether or not to do. In some assays, the senses used herein to describe cells or animals that have been engineered for altered expression of genes in the SREBP or interaction pathway, or other pathways associated with lipid metabolism and / or adipogenesis. Use the created genetic background.
[0033]
Cell-based assays
In cell-based assays, HEK-293 cells, CHO cells, primary hepatocytes, or McA-RH7777 (DeBose-Boyd et al., 2001, PNAS 98: 1477-1482) or HEPG2 (Kotzka et al., 2000 J. Lipid Res. 41) : 99-108), and various mammalian cell types capable of producing SREBP signals, including hepatocyte cell lines. Cell-based assays detect endogenous SREBP pathway activity and may be based on recombinant expression of SREBP pathway components. Cell-based assays typically use low cholesterol or low PUFA conditions, or culture conditions that allow SREBP signals such as glucose or insulin stimulation. Candidate modulators are typically added to the cell culture, but may be injected into the cells or delivered by any other effective means.
In certain embodiments, SREBP pathway activity is assessed by measuring the expression of a SREBP transcription target. Many transcription targets are known (eg, Osborne TF, 2001, J Biol Chem 275: 32379-32382; Horton JD et al., 1998, J Clin Invest 101: 2331-2339; Shimano H et al., 1997, J Clin Invest. 100: 2115-2124; Shimomura I et al., 1999, J Biol Chem 274: 30028-30032). Any available means for expression analysis may be used as previously described. Typically, mRNA expression is detected. In a preferred application, Taqman analysis is used to measure mRNA expression directly. Alternatively, expression is performed under the control of a control sequence (eg, an enhancer / promoter region) of a SREBP transcription target gene, a gene set including a sequence encoding a reporter gene (luciferase, GFP or other fluorescent protein, β-galactosidase, etc.). Monitored indirectly from the replacement reporter construct. Methods for making and using reporter constructs are well known (eg, Chakravarty K. et al., 2001 J. Biol. Chem. 276: 34816-34823).
[0034]
In other embodiments, the assay monitors SREBP processing phenomena, such as cleavage of the membrane-bound form of SREBP, or nuclear translocation or accumulation of the activated form of SREBP. These phenomena can be monitored directly by monitoring the level of membrane binding and the cleavage form of the protein. Typically, cells are fractionated and protein levels in the nuclear and membrane fractions are measured using immunohistology. Alternatively, SREBP cleavage can be monitored indirectly using a specific reporter for SREBP cleavage. In one example, a fusion construct comprising a sequence encoding a soluble catalytic domain in which a signal peptide and alkaline phosphatase (AP) are linked to the C-terminal (regulatory) domain of SREBP is introduced into cells. SREBP cleavage is monitored as secretion of AP, but detected using a standard alkaline phosphatase assay (Sakai J., et al., 1998, Mol. Cell 2: 505-514). In another example, a fusion construct is made and the transcriptional activator domain of SREBP is replaced by another transcriptional activator domain, such as yeast GAL4. The substituted domains are preferably from a different species and, when GAL4 is used, specifically activate transcription of the reporter gene under the control of response control sequences such as UAS.
In another embodiment, the assay measures the effect of the candidate modulator on the functional output of the SREBP signal, such as lipid accumulation and lipid metabolism. In one preferred application, lipid accumulation is measured by staining immobilized cells with Oil Red O (Foretz et al., 1999, PNAS 96: 12737-12742). In another preferred application, lipogenesis is monitored by measuring the incorporation of C14 acetate into either cholesterol or fatty acids (Pai, J-T et al., 1998, J. Biol. Chem. 273: 26138-26148).
[0035]
Animal assays
Various non-human animal models of lipid metabolism disorders are used to test candidate HisRS modulators. Such models are typically genetically engineered to mis-express (eg, overexpress or lack expression) genes involved in lipid metabolism, adipogenesis, and / or the SREBP pathway. Use a modified animal. In addition, certain dietary conditions and / or administration or certain biologically active compounds contribute or create animal models of lipid and / or metabolic disorders. Assays generally require systemic delivery of the candidate modulator by oral administration, injection (intravenous, subcutaneous, intraperitoneal), bolus administration, and the like.
In one embodiment, the assay uses a model mouse for diabetes and / or insulin resistance. Mice in which genes are knocked out in the leptin pathway, such as ob (leptin) or db (leptin receptor), or in the insulin signaling pathway, such as insulin receptor (InR) or insulin receptor substrate (IRS), show symptoms of diabetes, Lipid accumulation (fatty liver) and often show increased plasma lipid levels (Nishina et al., 1994, Metabolism 43: 549-553; Michael et al., 2000, Mol Cell 6: 87-97; Bruning JC et al. , 1998, Mol Cell 2: 559-569). Certain sensitive wild-type mice, such as C57BL / 6, exhibit similar symptoms when fed a high fat diet (Linton and Fazio, 2001, Current Opinion in Lipidology 12: 489-495). Accordingly, suitable assays using these models test whether administration of a candidate modulator alters, and preferably reduces, lipid accumulation in the liver. Lipid levels in plasma and adipocytes are also tested. Methods for assaying lipid content typically include FPLC or colorimetric assays (Shimano H et al., 1996, J Clin Invest 98: 1575-1584; Hasty et al., 2001, J Biol Chem 276: 37402-37408), and Lipid synthesis, such as scintillation measurement of incorporation of a radiolabeled substrate (Horton JD et al., 1999, J Clin Invest 103: 1067-1076), is well known in the art. Other useful assays test blood glucose levels, insulin levels, and insulin sensitivity (eg, Michael MD, 2000, Molecular Cell 6:87). In addition, SREBP pathway activity may be tested by examining the variability of transcription of the SREBP target gene in the liver. Typical target genes are involved in fatty acid metabolism, including acetyl-CoA carboxylase, fatty acid synthase, ATP citrate lyase, glycerol-3-phosphate acyltransferase, glucose-6-phosphate dehydrogenase, malic enzyme, and stearoyl-CoA desaturase. 1 (Shimomura I et al., 1999, supra). Other target genes are associated with cholesterol metabolism and include HMG-CoA synthesis, HMG-CoA reductase, squalene synthase, lipoprotein lipase, low density lipoprotein receptor (LDLR), and the like (Horton JD et al., 1998, supra).
[0036]
Other suitable animal models have specific alterations in the SREBP pathway gene. For example, mice overexpressing a constitutively active form of SREBP under the control of the PEPCK promoter develop fatty liver indication. In the background of low density lipoprotein receptor (LDLR) null, plasma lipids are further increased (Horton JD et al., 1999, J Clin Invest 103: 10677-1076). Assays using these mice measure both liver and plasma lipid levels.
In another embodiment, the assay uses a mouse model of lipoprotein biology and cardiovascular disease. For example, knockout mice for apolipoprotein E (apoE) show elevated plasma cholesterol and spontaneous arterial damage (Zhang SH, 1992, Science 258: 468-471). Transgenic mice that overexpress cholesterol ester transfer protein (CETP) also have increased plasma lipid levels (particularly very low density lipoprotein [VLDL] and low density lipoprotein [LDL] cholesterol levels) and plaque formation in arteries. (Marotti KR et al., 1993, Nature 364: 73-75). In assays using these models, administration of candidate modulators reduces plasma lipid levels by reducing preatherogenic LDL and VLDL levels, increasing HDL, or decreasing total lipid (including triglyceride) levels. Is tested to make changes. In addition, histological analysis of arterial morphology and lesion formation (ie, number and magnitude of lesions) indicates whether candidate modulators can reduce the progression and / or severity of atherosclerosis. Is shown. Many other mouse models for atherosclerosis are available, including the knockout of Apo-A1, PPARγ, and the scavenger receptor (SR) -B1 in the LDLR or ApoE-null background (eg, Glass CK and Witztum JL, 2001, Cell 104: 503-516).
In another embodiment, the ability of candidate modulators to alter plasma lipid levels and the progression of atherosclerosis is tested in a mouse model for multiple lipid disorders. For example, mice in which both the leptin and LDL receptor genes are knocked out show hypercholesterolemia, hypertriglyceridemia and arterial dysfunction, impaired fuel metabolism, increased plasma residual lipoprotein, diabetes, and atherogenicity. Provide a model of the association between arteriosclerosis (Hasty AH et al., 2001, supra).
[0037]
Diagnosis method
The discovery that HisRS is involved in the SREBP pathway, for the diagnostic and prognostic assessment of diseases and disorders associated with the SREBP pathway, and for identifying patients predisposed to such diseases and disorders Various methods are provided for use. As previously described, any method for assessing HisRS expression in a sample is used. For example, such methods may be as described above: (1) detecting the presence of a HisRS gene mutation, or detecting either over- or under-expression of HisRS mRNA compared to a non-disturbed condition; (2) non-disturbed HisRS oligonucleotides and antibodies to HisRS for the detection of over or under abundance of HisRS gene product compared to status; and (3) detection of perturbations or abnormalities in HisRS-mediated biological pathways And the like.
All sources, including patents, patent specifications, publications, and genes and sequences accessible through Genbank identification numbers and the indicated website are incorporated in their entirety.

Claims (30)

A method for identifying a candidate SREBP pathway modulator, said method comprising:
(A) providing an assay system comprising a HisRS polypeptide or nucleic acid;
(B) contacting the test agent with the assay system under conditions in which the system provides control activity in the absence of the test agent; and (c) between the activity deflected by the test agent and the control activity. Detecting a biased activity of the test agent in a screening assay system, wherein the difference identifies the test agent as a candidate for a SREBP pathway modulator. 2. The method of claim 1, wherein the assay system comprises a screening assay comprising a HisRS polypeptide, and wherein the candidate test agent is a small molecule modulator. 3. The method of claim 2, wherein said screening assay is an enzymatic assay. 2. The method of claim 1, wherein said assay system comprises a binding assay comprising a HisRS polypeptide, and wherein said candidate test agent is an antibody. 2. The method of claim 1, wherein said assay system comprises an expression assay comprising a HisRS nucleic acid, and wherein said candidate test agent is a nucleic acid modulator. The method according to claim 5, wherein the nucleic acid modulator is an antisense oligomer. 7. The method of claim 6, wherein said nucleic acid modulator is PMO. Wherein the assay system comprises cultured cells or non-human animals expressing HisRS, wherein the assay system comprises an assay for detecting an agent-biased change in SREBP pathway, lipid metabolism, or an activity associated with lipogenesis. Item 1. The method according to Item 1. 9. The method according to claim 8, wherein said assay system comprises cultured cells. 10. The method of claim 9, wherein said assay detects a phenomenon selected from the group consisting of expression of SREBP transcription target, processing of SREBP protein, lipid accumulation, and lipid metabolism. 9. The method of claim 8, wherein the second assay system comprises a non-human animal. The method according to claim 11, wherein the non-human animal is a mouse that provides a model for diabetes and / or insulin resistance. 12. The method of claim 11, wherein said non-human animal mis-expresses the SREBP pathway gene. The assay system is selected from the group consisting of hepatic lipid accumulation, plasma lipid accumulation, fatty lipid accumulation, blood glucose levels, insulin levels, and insulin sensitivity, and expression of SREBP transcription target. 14. The method according to claim 12 or 13, comprising an assay to detect. 12. The method of claim 11, wherein said non-human animal provides a model of atherosclerosis. 16. The method of claim 13 or claim 15, wherein the assay system comprises an assay that detects a phenomenon selected from the group consisting of plasma lipid levels and arterial lesion formation. 17. The method of claim 16, wherein said assay system detects plasma lipid levels, and wherein the detected lipid is triglyceride, cholesterol, or lipoprotein. (D) providing a second assay system comprising a cultured cell or a non-human animal expressing HisRS;
(E) contacting the test agent of (b) or an agent derived therefrom with a second assay system under conditions in which the system provides control activity in the absence of the test agent or agent derived therefrom. And (f) confirming that the difference between the activity biased by the agent of the second assay system and the activity of the control is that the test agent or an agent derived therefrom is a candidate SREBP pathway modulator; The second assay system includes a second assay that detects a drug-biased change in an activity associated with the SREBP pathway, lipid metabolism, or adipogenesis. Detect:
The method of claim 1, comprising a further step. 19. The method of claim 18, wherein the second assay system comprises a cultured cell. 20. The method of claim 19, wherein the second assay detects a phenomenon selected from the group consisting of SREBP transcription target expression, SREBP protein processing, lipid accumulation and lipid metabolism. 18. The method of claim 17, wherein the second assay system comprises a non-human animal. 22. The method according to claim 21, wherein the non-human animal is a mouse that provides a model for diabetes and / or insulin resistance. 22. The method of claim 21, wherein said non-human animal mis-expresses the SREBP pathway gene. The second assay system is selected from the group consisting of hepatic lipid accumulation, plasma lipid accumulation, fatty lipid accumulation, blood glucose levels, insulin levels and insulin sensitivity, and expression of SREBP transcription target. 24. The method according to claim 22 or 23, comprising an assay for detecting a phenomenon. 22. The method of claim 21, wherein said non-human animal provides a model of atherosclerosis. 26. The method of claim 23 or 25, wherein the assay system comprises an assay that detects a phenomenon selected from the group consisting of plasma lipid levels and arterial lesion formation. A method of modulating SREBP pathway activity in a mammalian cell, comprising contacting the cell with an agent that specifically binds a HisRS polypeptide or nucleic acid. 28. The method of claim 27, wherein the agent is administered to a mammal predicted to exhibit a pathology associated with the SREBP pathway. 28. The method of claim 27, wherein said agent is a small molecule modulator, a nucleic acid modulator, or an antibody. 28. The method of claim 27, wherein SREBP pathway activity is reduced.