WO1998049555A1 - Methods for identifying ligands for nuclear hormone receptors - Google Patents
Methods for identifying ligands for nuclear hormone receptors Download PDFInfo
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- WO1998049555A1 WO1998049555A1 PCT/US1998/006446 US9806446W WO9849555A1 WO 1998049555 A1 WO1998049555 A1 WO 1998049555A1 US 9806446 W US9806446 W US 9806446W WO 9849555 A1 WO9849555 A1 WO 9849555A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
Definitions
- the present invention relates to methods for the modulation of nuclear receptor mediated processes .
- the present invention relates to methods for the identification of compounds useful for modulation of such processes.
- the present invention relates to methods for the identification of ligands for the PPARs.
- the present invention relates to methods for monitoring fatty acid- containing foodstuffs for the presence of beneficial fatty acids therein.
- the present invention relates to the use of a specific class of compounds for the modulation of processes mediated by peroxisome proliferator activated receptor-alpha (PPAR- ⁇ .) .
- the present invention relates to the use of a specific class of compounds for the modulation of processes mediated by peroxisome proliferator activated receptor-delta (PPAR- ⁇ ) .
- the present invention relates to the use of a specific class of compounds for the modulation of processes mediated by peroxisome proliferator activated receptor-gamma (PPAR- ⁇ ) .
- the present invention relates to methods to induce fatty acid degradation in a subject.
- FAs Fatty acids
- Fatty acids are ubiquitous biological molecules that are utilized as metabolic fuels, as covalent regulators of signaling molecules and as essential components of cellular membranes. It is thus logical that FA levels should be closely regulated. Indeed, some of the most common medical disorders in industrialized societies (e.g., cardiovascular disease, hyperlipidemia, obesity and insulin resistance) are characterized by altered levels of FAs or their metabolites (see, for example, Durrington, in Postgrad Med J 69 Suppl 1, S18-25; discussion S25-9 (1993) and Reaven, in J Intern Med Suppl 736, 13-22 (1994)).
- PPAR ⁇ has been identified as a vertebrate nuclear hormone receptor which regulates genes involved in FA degradation ( ⁇ - and ⁇ -oxidation; see Schoonjans et al., in Biochim Biophvs Acta 1302:93-109 (1996)). PPAR ⁇ . is highly expressed in the liver and was originally identified by Green and colleagues as a molecule that mediates the transcriptional effects of drugs that induce peroxisome proliferation in rodents (see Issemann & Green in Nature 347:645-50 (1990)). Mice lacking functional PPAR ⁇ . are incapable of responding to these agents and fail to induce expression of a variety of genes required for the metabolism of FAs in peroxisomes, mitochondria and other cellular compartments (see Lee et al . , in Mol Cell Biol
- PPAR ⁇ is a member of the nuclear receptor superfamily, which includes receptors for the steroid, thyroid and retinoid hormones (see Mangelsdorf & Evans in Cell 83:841-50 (1995)).
- Two other PPAR ⁇ .-related genes (PPAR ⁇ and PPAR ⁇ ) have been identified in mammals.
- PPAR ⁇ is highly enriched in adipocytes, while the ⁇ isoform is ubiquitously expressed (see Schoonjans et al., supra) .
- all of the PPAR isoforms contain a central DNA binding domain that recognizes response elements in the promoters of their target genes.
- PPAR response elements are composed of a directly repeating core-site separated by 1 nucleotide (see Kliewer et al., in Nature 358:771-4 (1992)). In order to recognize a PPRE, PPARs must heterodimerize with the 9- cis retinoic acid receptor (RXR) .
- RXR 9- cis retinoic acid receptor
- PPARs activate transcription through a conserved C-terminal ligand binding domain.
- sequence analysis indicates that its C-terminal region is similar to the ligand binding domains of known members of the nuclear hormone receptor superfamily. This has prompted an intense search for the identification of ligands for the PPARs.
- 15-deoxy- ⁇ 12,14 - prostaglandin J 2 (15d-J 2 ) has been identified as a ligand for PPAR ⁇ (see, for example, Forman et al . , in Cell 83:803- 12 (1995) and Kliewer et al . , in Cell 83:813-9 (1995)).
- thiazolidinediones promotes differentiation of pre-adipocytes into mature, triglyceride-containing fat cells.
- thiazolidinediones have been shown to increase body weight in animals (see Zhang et al . , in J Biol Chem 271:9455-9 (1996) ) , suggesting that 15d-J 2 may be utilized as an in vivo signal to store FAs in the form of triglycerides .
- PPAR ⁇ appears to regulate FA oxidation, suggesting that PPAR ⁇ ligands may represent endogenous signals for FA degradation (see Schoonjans et al . , supra) .
- Green and colleagues originally demonstrated that PPAR ⁇ is activated by fibrates (see Issemann & Green, supra) , a group of drugs that induce peroxisome proliferation and FA oxidation in rodents.
- Figure 1A presents the chemical structures of some compounds that are demonstrated herein to be ligands for PPAR ⁇ or ⁇ .
- Figure IB presents data demonstrating that fibrates selectively activate PPAR ⁇ in a cell-based transient transfection assay. Cells were treated with the following concentrations of each compound: 5 ⁇ M y 14,643, 300 ⁇ M ciprofibrate, 300 ⁇ M clofibrate and 1 ⁇ M BRL 49653.
- Figure 1C presents a comparison of the dose response profile of wild-type PPAR ⁇ (solid circles) with PPAR ⁇ -G (i.e., a mutant wherein the Glu at position 282 is replaced with Gly; see Hsu et al . , in Mol Pharmacol 48:559- 67 (1995) ,- represented in the figure with open circles) in the transient transfection assay (left panel) and the ligand induced c.omplexation (LIC) assay (right panel; see also Example 4) .
- the ligand induced complex was quantified by phosphorimaging analysis.
- Ligand induced binding represents the amount of complex produced at any concentration of ligand, minus that produced in the absence of ligand. Maximal induced binding was defined to be 100%; binding observed at other concentrations was normalized to this value.
- Figure 2A illustrates the activation of PPAR ⁇ by
- Figure 2B illustrates the enhancement of PPAR ⁇ - RXR ⁇ heterodimer formation by FAs and fatty alcohols. All compounds were added to a final concentration of 30 ⁇ M except for Wy 14,643 which was added to a final concentration of 5 ⁇ M . Saturated FAs and alcohols are indicated by their chain length.
- Unsaturated FAs are as follows: linoleic acid (cis- ⁇ 0,12 -Cl8 :2) , ⁇ -linolenic acid (cis- ⁇ 9 ' 1 ,15 -Cl8 :3) , ⁇ -linolenic acid (cis- ⁇ 6 ' 0,1 -Cl8 :3) , arachidonic acid (cis- ⁇ 5 ' 8 - 11 ' 1 -C20 :4) , erucic acid (cis- ⁇ 13 -C22 : 1) and nervonic acid (cis- ⁇ 15 -C24 :1) .
- Figure 2C illustrates that inhibitors of ⁇ -oxidation both activate (left panel) and bind (right panel) to PPAR ⁇ . Experiments were performed as described with respect to Figure 1 (see also, Example 6) .
- Triacsin C (10 ⁇ M , left panel; 30 ⁇ M , right panel) was used as an inhibitor of fatty LC-FACS.
- Inhibitors of carnitine palmitoyltransferase I included LY 171883 (30 ⁇ M) , 2-bromopalmitate (2Br-C16; 5 ⁇ M) and tetradecylglycidic acid (TDGA, 5 ⁇ M) .
- Fatty acyl-CoA dehydrogenase was inhibited with octylthioproprionic acid (OTP, 30 ⁇ M) , tetradecylthioproprionic acid (TTP, 30 ⁇ M) , nonylthioacetic acid (NTA, 30 ⁇ M) and tetradecylthioacetic acid (TTA, 30 ⁇ M) .
- OTP octylthioproprionic acid
- TTP tetradecylthioproprionic acid
- NTA nonylthioacetic acid
- TTA tetradecylthioacetic acid
- Figure 3A relates to the identification of eicosanoid ligands for PPAR ⁇ .
- carbaprostacyclin (cPGI) iloprost, 8-hydroxyeicosatetraenoic acid (8S-HETE) and 8-hydroxyeicosapentaenoic acid (8S-HEPE) are seen to transactivate (left panel) and bind (right panel) to PPAR ⁇ .
- Figure 3B presents dose response curves comparing the potency of 8S-HETE (solid circles) , cPGI (triangles) and Wy 14,643 (shaded squares) in the transactivation of (left panel) and binding to (right panel) PPAR ⁇ .
- Figure 4A demonstrates that PPAR ⁇ , PPAR ⁇ and
- PPAR ⁇ display distinct ligand response profiles.
- linoleic acid, arachidonic acid, cPGI and iloprost are seen to transactivate (left panel) and bind to (right panel) PPAR ⁇ .
- compounds were added to test cells at the following concentrations: 5 ⁇ M Wy 14,643; 100 ⁇ M ciprofibrate; 1000 ⁇ M clofibrate; 5 ⁇ M BRL 49653; 30 ⁇ M C12, C16, linoleic acid, ⁇ -linoleic, arachidonic, docosahexaenoic (DHA, all-Z- ⁇ ' 7 ' 10 ' 13 ' 16 ' 19 -C22 :6) and eicosapentaenoic (EPA, all-Z- ⁇ 5 ' 8 ' 11 ' 14 ' 17 -C20 :5) acids; 5 ⁇ M 2Br-C16; 30 ⁇ M TTA; 10
- Figure 4B presents a comparison of the responsiveness of PPAR ⁇ (solid bars) , PPAR ⁇ (shaded bars) and PPAR ⁇ (striped bars) to various compounds.
- Control cells i.e., containing reporter vector only
- spotted bars After transfection, cells were treated with the following concentrations of compounds: 30 ⁇ M C16; 5 ⁇ M 2Br-C16; 30 ⁇ M TTA, linoleic, arachidonic acids and EPA; 3 ⁇ M ⁇ 8-HETE; 10 ⁇ M PGA,; 3 ⁇ M 15d-PGJ 2 ; 1 ⁇ M cPGI; 5 ⁇ M Wy 14,643 and BRL 49653.
- invention methods comprise: determining the binding activity of a homodimer or heterodimer containing said member with respect to a hormone response element (HRE) in the presence of said test compound, relative to the binding activity of said homodimer or heterodimer with respect to said HRE in the absence of said test compound.
- HRE hormone response element
- the invention method to determine if test compounds are ligands for members of the nuclear receptor superfamily comprises: contacting said member and a dimeric partner therefor with said test compound, and monitoring for the formation of a complex comprising said member, said dimeric partner therefor and said test compound, wherein formation of said complex indicates that said test compound is a ligand for said member.
- the phrase "members of the nuclear receptor superfamily” refers to hormone binding proteins that operate as ligand-dependent transcription factors, including identified members of the steroid/thyroid superfamily of receptors for which specific ligands have not yet been identified (referred to hereinafter as "orphan receptors") . These hormone binding proteins have the intrinsic ability to bind to specific DNA sequences. Following binding, the transcriptional activity of target gene (i.e., a gene associated with the specific DNA sequence) is modulated as a function of the ligand bound to the receptor.
- target gene i.e., a gene associated with the specific DNA sequence
- DNA-binding domains of all of these nuclear receptors are related, consisting of 66-68 amino acid residues, and possessing about 20 invariant amino acid residues, including nine cysteines.
- a member of the superfamily can be identified as a protein which contains the above-mentioned invariant amino acid residues, which are part of the DNA-binding domain of such known steroid receptors as the human glucocorticoid receptor (amino acids 421-486) , the estrogen receptor (amino acids 185-250) , the mineralocorticoid receptor (amino acids 603-668) , the human retinoic acid receptor (amino acids 88-153) .
- the highly conserved amino acids of the DNA-binding domain of members of the superfamily are as follows: Cys - X - X - Cys - X - X - Asp* - X -
- X designates non-conserved amino acids within the DNA-binding domain; the amino acid residues denoted with an asterisk are residues that are almost universally conserved, but for which variations have been found in some identified hormone receptors; and the residues enclosed in parenthesis are optional residues (thus, the DNA-binding domain is a minimum of 66 amino acids in length, but can contain several additional residues) .
- Exemplary members of the steroid/thyroid superfamily of receptors include steroid receptors such as glucocorticoid receptor, mineralocorticoid receptor, progesterone receptor, androgen receptor, vitamin D 3 receptor, and the like; plus retinoid receptors, such as RAR ⁇ , RAR ⁇ , RAR ⁇ , and the like, plus RXR ⁇ , RXR ⁇ , RXR ⁇ , and the like; thyroid receptors, such as TR ⁇ , TR ⁇ , and the like; as well as other gene products which, by their structure and properties, are considered to be members of the superfamily, as defined hereinabove.
- steroid receptors such as glucocorticoid receptor, mineralocorticoid receptor, progesterone receptor, androgen receptor, vitamin D 3 receptor, and the like
- retinoid receptors such as RAR ⁇ , RAR ⁇ , RAR ⁇ , and the like, plus RXR ⁇ , RXR ⁇ , RXR ⁇ , and the like
- thyroid receptors such
- orphan receptors examples include the PPARs (e.g., PPAR ⁇ , PPAR ⁇ and PPAR ⁇ ), HNF4 [see, for example, Sladek et al . , in Genes & Development 4: 2353-2365 (1990)], the COUP family of receptors [see, for example, Miyajima et al . , in Nucleic Acids Research 16 : 11057-11074 (1988), Wang et al . , in Nature 340: 163-166 (1989)], COUP-like receptors and COUP homologs, such as those described by Mlodzik et al . , in Cell 0: 211-224 (1990) and Ladias et al . , in Science 251: 561-565 (1991) , the ultraspiracle receptor [see, for example, Oro et al . , in Nature 347: 298-301 (1990)], and the like.
- PPARs e.g.
- RAR retinoic acid receptor
- T 3 R thyroid hormone receptor
- VDR vitamin D 3 receptor
- PPAR retinoid X
- RXR 9 - cis retinoic acid receptor
- Naturally occurring HREs are composed of direct repeats (i.e., DRs; see Umesono et al . , in Cell 65 -.1255- 1266 (1991), inverted repeats (i.e., IRs; see Umesono et al., in Nature 336:262-265 (1988), and Williams et al . in
- X n sequence also serves as a gap which separates the two core-binding sites.
- spacers of 1, 3, 4 and 5 nucleotides serve as preferred response elements for heterodimers of RXR with PPAR, VDR, T 3 R and RAR, respectively (see, for example, Naar et al . , in Cell Little:1267-1279 (1991); Umesono et al .
- the optimal gap length for each heterodimer is determined by protein-protein contacts which appropriately position the DNA binding domains (DBDs) of RXR and its partner (see, for example, Kurokawa et al . , in Genes Dev. 2:1423-1435 (1993); Perlmann et al . , in Genes Dev. 7:1411- 1422 (1993); Towers et al . , in Proc. Natl. Acad. Sci . USA 10:6310-6314 (1993); and Zechel et al . , in EMBO J. 13:14- 1424 (1994) ) .
- DBDs DNA binding domains
- Direct repeat hormone response elements contemplated for use in the practice of the present invention are composed of at least one direct repeat of two or more half sites, optionally separated by one or more spacer nucleotides (with spacers of 1-5 preferred) .
- the spacer nucleotides can be selected from any one of A, C, G or T.
- Each half site of direct repeat HREs contemplated for use in the practice of the invention comprises the sequence
- R is selected from A or G
- M is selected from A or C; with the proviso that at least 4 nucleotides of said -RGBNNM- sequence are identical with the nucleotides at corresponding positions of the sequence -AGGTCA- .
- Response elements employed in the practice of the present invention can optionally be preceded by N ⁇ , wherein x falls in the range of 0 up to 5.
- PPAR response elements contemplated for use in the practice of the present invention are composed of at least one direct repeat of two or more of the above- described half sites, separated by a spacer of one nucleotide.
- the spacer nucleotide can be selected from any one of A, C, G or T.
- Presently preferred PPREs contain at least one copy (with one, two or three copies most common) of the minimal sequence:
- AGGACA A AGGTCA (SEQ ID NO :2) .
- lipomodulatory agents e.g., fibrates
- optionally substituted long-chain mono-, di- or polycarboxylic acid containing at least one site of unsaturation such as, for example, monounsaturated fatty acids (e.g., erucic acid, nervonic acid, and the like), polyunsaturated fatty acids (e.g., linoleic acid, ⁇ -linolenic acid, ⁇ -linolenic acid, arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid, and the like) , eicosanoids (e.g., arachidonic acid, prostaglandin A,, prostaglandin A 2 , prostaglandin B 2 , prostaglandin J 2 , 8, 9-d
- invention methods comprise: determining the binding activity of a PPAR ⁇ - containing heterodimer with respect to a PPAR response element (PPRE) in the presence of the fatty acid(s) contained in said foodstuff, relative to the binding activity of said PPAR ⁇ - containing heterodimer with respect to said PPRE in the absence of said fatty acid(s) , wherein binding of said heterodimer to said PPRE is indicative of the presence of beneficial fatty acids in said foodstuff.
- PPRE PPAR response element
- the invention method comprises: determining the ability of the fatty acid(s) contained in said foodstuff to activate a PPAR ⁇ - responsive reporter in an assay system comprising:
- PPAR ⁇ PPAR ⁇ , RXR ⁇ , and a PPAR ⁇ -responsive reporter comprising a PPAR response element (PPRE) in operative communication with a reporter gene, wherein expression of the reporter gene product is indicative of the presence of beneficial fatty acids in said foodstuff.
- PPRE PPAR response element
- fatty acids in a foodstuff can be separated therefrom using standard techniques, such as, for example, by extraction.
- methods to characterize the profile of fatty acids in a fatty acid- containing foodstuff are provided.
- invention methods comprise: determining the quantity of binding of a PPAR ⁇ - containing heterodimer, a PPAR ⁇ -containing heterodimer or a PPAR ⁇ -containing heterodimer, to a PPAR response element (PPRE) in the presence of the fatty acid(s) contained in said foodstuff, relative to the quantity of binding of said PPAR ⁇ - containing heterodimer, said PPAR ⁇ - containing heterodimer or said PPAR ⁇ -containing heterodimer, respectively, to said PPRE in the absence of the fatty acid(s) contained in said foodstuff.
- PPRE PPAR response element
- invention methods comprise: determining the ability of the fatty acid(s) contained in said foodstuff to activate a PPAR ⁇ - responsive reporter, a PPAR ⁇ -responsive reporter or a PPAR ⁇ -responsive reporter, in an assay system comprising:
- PPAR ⁇ PPAR ⁇
- PPAR ⁇ PPAR ⁇
- PPAR ⁇ PPAR ⁇
- PPAR ⁇ PPAR ⁇
- PPAR ⁇ PPAR ⁇
- PPAR ⁇ PPAR ⁇
- PPAR ⁇ PPAR ⁇
- PPAR ⁇ PPAR ⁇
- PPAR ⁇ PPAR ⁇
- PPAR ⁇ PPAR ⁇
- PPAR ⁇ PPAR ⁇
- PPAR ⁇ PPAR ⁇
- PPAR ⁇ PPAR ⁇
- PPAR ⁇ PPAR ⁇
- PPAR ⁇ PPAR ⁇
- PPAR ⁇ PPAR ⁇
- the activity profile of the fatty acid content of food can readily be obtained, so that a given foodstuff can be characterized in terms of its ability to activate PPAR ⁇ - mediated pathways (e.g., fatty acid metabolism), PPAR ⁇ - mediated pathways and/or PPAR ⁇ -mediated pathways (e.g., fatty acid storage) .
- PPAR ⁇ - mediated pathways e.g., fatty acid metabolism
- PPAR ⁇ - mediated pathways e.g., fatty acid storage
- various profiles may be indicated for actual consumption.
- methods to induce fatty acid degradation in a subject comprising administering to a subject an effective amount of a PPAR ⁇ ligand.
- PPAR ⁇ induces transcription of a number of gene products that contribute to the metabolism of FAs . These include enzymes necessary for the degradation of FAs through ⁇ - and ⁇ - oxidation pathways. It has long been established that metabolic intermediates modulate feedback control by promoting allosteric changes in enzymatic activity. The demonstration that FAs bind to PPAR ⁇ provides direct evidence that metabolic intermediates can also regulate transcription. This complements the immediate effects of allosteric control by modulating the metabolic capacities of the organism over longer time periods .
- PPAR ⁇ can recognize a broad array of ligands . This is unique among the nuclear receptors and suggests that PPAR ⁇ senses broad changes in FA status and dietary inputs. In particular, as metabolism may vary from cell-to-cell and tissue-to-tissue, PPAR ⁇ may act locally to integrate a variety of cell-specific metabolic parameters. In contrast to PPAR ⁇ (which promotes FA catabolism) , PPAR ⁇ appears to stimulate the opposing function of FA storage. The results presented herein demonstrate that PPAR ⁇ ligands are distinct from those of PPAR ⁇ . The ability of these receptors to respond to distinct metabolic cues provides a potential mechanism for the animal to maintain a balance between FA breakdown and storage.
- 8S-HETE is a high affinity ligand for PPAR ⁇ .
- the identification of this ligand in the skin suggests that it may play a specialized function in this tissue.
- other eicosanoids were found which activate but fail to bind to PPAR (e.g. PGA, and PPAR ⁇ ) (see Yu et al., in J Biol Chem 270:23975-83 (1995) and Figs. 3A and 4A) .
- LTB 4 binds xenopus PPAR ⁇ with an affinity of approximately 100 nM (see Devchand et al . , in Nature 384:39-43 (1996)). However, non-specific binding to PPAR ⁇ was not accounted for and half-maximal displacement required 10-50 ⁇ M of unlabeled LTB 4 . Since neither activation nor binding were detected with 10 ⁇ M LTB 4 (see Fig. 3A) , it is unclear whether LTB 4 is a physiologically relevant ligand for mouse PPAR ⁇ .
- dietary PUFAs can be beneficial in this regard (see, for example, Willumsen et al., in Lipids 28:683-90 (1993) and Spady et al . , in Annu Rev Nutr 13:355- 81 (1993)). This may reflect both activation of PPAR- regulated ⁇ - and ⁇ -oxidation pathways (see Green in Mutat Res 333:101-9 (1995)) as well as PUFA-dependent suppression of lipogenic and glycolytic enzymes (see Jump et al., in J Lipid Res 35:1076-84 (1994)).
- a negative PUFA response element has been identified in the promoter of the pyruvate kinase gene (see Liimatta et al . , in Mol Endocrinol 8:1147-53 (1994)).
- This response element binds HNF-4, a constitutively active orphan nuclear receptor whose DNA-binding specificity overlaps that of PPAR-RXR heterodimers . It has previously been shown that PPAR ⁇ antagonizes HNF-4 by down-regulating its expression in liver and by binding non-productively to HNF-4 response elements (see Hertz et al . , in J Biol Chem 271:218-24 (1996)).
- drugs of the fibrate class are also known to regulate transcription of apolipoproteins A- 1, A-11 and C-III (see Schoonjans et al . , supra) and are useful for the treatment of hyperlipidemias.
- the effective doses of the best available drugs are in the high micromolar range.
- the demonstration herein that fibrates bind directly to PPAR ⁇ indicates that screening for high affinity PPAR ⁇ ligands employing the methods described herein provides a rapid approach for the development of more effective treatments for these lipid- related disorders. Since PPAR isoforms have distinct functions, the relative specificity of a drug for each PPAR isoform may be an important factor in evaluating its therapeutic potential.
- inventions to modulate expression of PPAR ⁇ -responsive genes in a biological system.
- invention methods comprise contacting a biological system with an effective amount of a lipomodulatory agent, an optionally substituted long-chain mono-, di- or polycarboxylic acid containing at least one site of unsaturation, and the like.
- modulate refers to the ability of a modulator for a member of the steroid/thyroid superfamily to either directly (by binding to the receptor as a ligand) or indirectly (as a precursor for a ligand or an inducer which promotes production of ligand from a precursor) induce expression of gene(s) maintained under hormone expression control, or to repress expression of gene(s) maintained under such control.
- PPAR ⁇ -responsive genes refers to genes whose expression products are involved in the biological, physiological, endocrinological, and other bodily processes which are mediated by receptor or receptor combinations which are responsive to the PPAR ⁇ ligands described herein (e.g., genes involved in fatty acid metabolism in peroxisomes, mitochondria and other cellular compartments (including FA degradation (by ⁇ - and ⁇ -oxidation) , and the like) . Modulation of such processes can be accomplished in vitro or in vivo. In vivo modulation can be carried out in a wide range of subjects, such as, for example, humans, rodents, sheep, pigs, cows, and the like.
- biological system refers to an intact organism or a cell-based system containing the various components required for response to the ligands described herein, e.g., an isoform of PPAR (i.e., PPAR ⁇ , PPAR ⁇ or PPAR ⁇ ), a silent partner for the PPAR isoform (e.g., RXR ⁇ ), and a PPAR-responsive reporter (which typically comprises a PPAR response element (PPRE) in operative communication with a reporter gene; suitable reporters include luciferase, chloramphenicol transferase, ⁇ -galactosidase, and the like) .
- PPAR i.e., PPAR ⁇ , PPAR ⁇ or PPAR ⁇
- RXR ⁇ a silent partner for the PPAR isoform
- a PPAR-responsive reporter which typically comprises a PPAR response element (PPRE) in operative communication with a reporter gene; suitable reporters include luciferase, chloramphenicol
- the phrase "effective amount” refers to levels of compound sufficient to provide circulating concentrations high enough to modulate the expression of an isoform of PPAR. Such a concentration typically falls in the range of about 10 nM up to 2 ⁇ M; with concentrations in the range of about 100 nM up to 500 nM being preferred. Since the activity of different compounds described herein may vary considerably, and since individual subjects may present a wide variation in severity of symptoms, it is up to the practitioner to determine a subject's response to treatment and vary the dosages accordingly.
- the above-described biologically active compounds can be administered in a variety of forms (e.g., in combination with a pharmaceutically acceptable carrier therefor) and by a variety of modes of delivery.
- exemplary pharmaceutically acceptable carriers include carriers suitable for oral, intravenous, subcutaneous, intramuscular, intracutaneous, and the like administration. Administration in the form of creams, lotions, tablets, dispersible powders, granules, syrups, elixirs, sterile aqueous or non-aqueous solutions, suspensions or emulsions, and the like, is contemplated.
- suitable carriers include emulsions, solutions, suspensions, syrups, and the like, optionally containing additives such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents, and the like.
- suitable carriers include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
- non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
- Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions . They can also be manufactured in the form of sterile water, or some other sterile injectable medium immediately before use.
- inventions to modulate expression of PPAR ⁇ -responsive genes in a biological system.
- invention methods comprise contacting a biological system with an effective amount of a lipomodulatory agent, an optionally substituted long-chain mono-, di- or polycarboxylic acid containing at least one site of unsaturation, and the like.
- PPAR ⁇ -responsive genes refers to genes whose expression products are involved in the biological, physiological, endocrinological, and other bodily processes which are mediated by receptor or receptor combinations which are responsive to the PPAR ⁇ ligands described herein. Modulation of such processes can be accomplished in vitro or in vivo . In vivo modulation can be carried out in a wide range of subjects, such as, for example, humans, rodents, sheep, pigs, cows, and the like.
- inventions to modulate expression of PPAR ⁇ -responsive genes in a biological system.
- invention methods comprise contacting a biological system with an effective amount of a lipomodulatory agent, an optionally substituted long-chain mono-, di- or polycarboxylic acid containing at least one site of unsaturation, and the like.
- PPAR ⁇ -responsive genes refers to genes whose expression products are involved in the biological, physiological, endocrinological, and other bodily processes which are mediated by receptor or receptor combinations which are responsive to the PPAR- ⁇ ligands described herein (e.g., cell differentiation to produce lipid-accumulating cells, regulation of insulin-sensitivity and blood glucose levels, especially as related to hypoglycemia/hyperinsulinism (resulting, for example, from abnormal pancreatic beta-cell function, insulin-secreting tumors and/or autoimmune hypoglycemia due to autoantibodies to insulin, the insulin receptor or autoantibodies that are stimulatory to pancreatic beta-cells) , the formation of macrophages which lead to the development of atherosclerotic plaques, and the like) . Modulation of such processes can be accomplished in vitro or in vivo. In vivo modulation can be carried out in a wide range of subjects, such as, for example, humans, rodents, sheep, pigs,
- CV-1 cells were grown and transfected as described by Forman et al . , in Cell 83:803-12 (1995) .
- the reporter construct, PPREx3 TK-LUC contained 3 copies of the acyl CoA oxidase PPRE upstream of the Herpes virus thymidine kinase promoter (see Kliewer et al . , in Nature 358:771-4 (1992)).
- Expression vectors contained the cytomegalovirus IE promoter/enhancer (pCMX) upstream of wild-type mouse PPAR ⁇ , mouse PPAR ⁇ l- ⁇ N (Met 105 -Tyr 475 ) , mouse PPAR ⁇ - ⁇ N (Leu 69 -Tyr 440 ) , mouse PPAR ⁇ -G (Glu 82 -> Gly) (see Hsu et al., in Mol Pharmacol 48:559-67 (1995)) or E. coli ⁇ - galactosidase as an internal control. Cells were exposed to the compounds for 24 hours then harvested and assayed for luciferase and ⁇ -galactosidase activity. All points were performed in triplicate and varied by less than 10%. Normalized luciferase activity was determined and plotted as fold-activation relative to untreated cells. Each experiment was repeated three or more times with similar results .
- pCMX cytomegalovirus IE promoter/enhancer
- CV-1 cells were transiently transfected with a PPAR responsive reporter, PPAR expression vectors and then treated with various hypolipidemic agents (Fig. IB).
- Wy 14,643 and BRL 49653 were included as positive controls since these compounds selectively activate PPAR ⁇ and ⁇ , respectively (see Forman et al., in Cell 83:803-12 (1995), Kliewer et al . , in Cell 83:813-9 (1995) and Kliewer et al . , in Proc Natl Acad Sci USA 91:7355-9 (1994) ) .
- fibrates are observed to selectively promote binding of PPAR ⁇ -RXR ⁇ heterodimers to labeled DNA in an electrophoretic mobility shift assay.
- Compounds were added at the following concentrations: 5 ⁇ M Wy 14,643, 100 ⁇ M ciprofibrate, 1000 ⁇ M clofibrate, 1 ⁇ M BRL 49653 and 1 ⁇ M LG268. Where excess receptor is employed, the amounts of PPAR ⁇ and RXR ⁇ are increased to 0.6 ⁇ l and 0.5 ⁇ l , respectively. When included, 1 ⁇ l of antibody is added to the reaction.
- PPAR ⁇ -activators This enhancement is unique to PPAR ⁇ -activators; enhanced binding was not observed with PPAR ⁇ -specific ligands such as BRL 49653, pioglitazone and troglitazone or the RXR- specific ligands LG268, LG69 and 9-cis retinoic acid.
- PPAR ⁇ and RXR ⁇ are verified to be components of the ligand induced complex by the observations that the complex is supershifted by PPAR ⁇ -specific and RXR ⁇ -specific antibodies, but not by pre-immune serum.
- epitope-tagged PPAR ⁇ is supershifted by an epitope-specific monoclonal antibody (12CA5) .
- the dose response profiles of wild-type PPAR ⁇ were compared to that of a previously characterized point mutant (PPAR ⁇ -G; see Hsu et al., in Mol Pharmacol 48:559-67 (1995)) that exhibits a decreased potency for PPAR ⁇ activators in co- transfection experiments.
- concentration required for half-maximal transcriptional activation by Wy 14,643 was 4-fold greater with the mutant receptor (Fig. IC, left panel) .
- phosphorimaging analysis revealed a similar increase in the amount of Wy 14,643 required for half-maximal ligand induced binding (LIC 50 ) with the mutant receptor (Fig. IC, right panel) .
- the LIC 50 for Wy 14,643 (600 nM) appears to provide an effective estimate of the actual dissociation constant.
- the LIC assay was utilized to determine which, if any, naturally occurring FAs bind to PPAR ⁇ at physiologic concentrations.
- the total concentration of non-esterified FAs in serum is approximately 700 ⁇ M (see Groop et al . , in Am J Physiol 263:E79-84 (1992)).
- Abundant dietary FAs such as linoleic and arachidonic acid have average concentrations of 25-30 ⁇ M and may reach much higher levels .
- the intracellular concentrations of these compounds are more difficult to determine but can be inferred from the Michaelis constant of long-chain fatty-acyl CoA s_ynthetase (LC-FACS, 20 ⁇ M; see Tanaka et al . , in Eur J Biochem 98:165-72 (1979)).
- LC-FACS 20 ⁇ M
- Tanaka et al . in Eur J Biochem 98:165-72 (1979)
- PUFAs The ability of PUFAs to bind to PPAR ⁇ was examined next. It was found that linoleic, ⁇ -linolenic, ⁇ -linolenic, arachidonic (Fig. 2A-B, right panel) , docosahexaenoic and eicosapentaenoic acids all bind to and activate PPAR ⁇ . In contrast, very-long-chain unsaturated FAs such as erucic and nervonic acids failed to bind or activate PPAR ⁇ (Fig. 2A-B, right panel) . This structure- activity relationship suggests that PPAR ⁇ ligands can be broadly defined as long-chain monocarboxylic acids. Optimal binding activity is observed with compounds containing a 16-20 carbon chain length with several double bonds in the chain.
- LC-FACS catalyzes the first step in the mitochondrial ⁇ -oxidation cascade (Fig. 2C, left panel) .
- Several groups have shown that inhibitors of subsequent steps in this pathway lead to activation of PPAR ⁇ and peroxisome proliferation (see Gottlich et al . , in Biochem Pharmacol 46:2177-84 (1993), Gulick et al . , in Proc Natl Acad Sci USA 91:11012-6 (1994), and Asiedu et al . , in Biochim Biophys Acta 1166:73-6 (1993)).
- This has contributed to the "lipid-overload" hypothesis which suggests that these inhibitors activate PPAR ⁇ by promoting the accumulation of an endogenous ligand.
- these enzymatic inhibitors are structural analogs of long- chain FAs, the possibility that they might also be PPAR ⁇ ligands was addressed.
- inhibitors of carnitine palmitoyltransferase I e.g., LY 171883, 2-bromopalmitate (2Br-C16) , and tetradecylglycidic acid (TDGA) ; see Foxworthy & Eacho, in Biochem J 252:409-14 (1988), Brady et al . , in Biochem J 241:751-7 (1987) and Kiorpes et al .
- Example 7 PPARs are Nuclear Eicosanoid Receptors
- 8S-HETE and cPGI activate with half-maximal activity at 200 nM and 2 ⁇ M, respectively (Fig. 3B, left panel) and bind PPAR ⁇ with affinities estimated to be 100 nM and 500 nM, respectively (Fig. 3B, right panel) .
- the naturally occurring 8S-HETE is the highest affinity ligand yet to be identified for PPAR ⁇ .
- PPAR ⁇ -RXR ⁇ heterodimers were formed in the presence of Wy 14,643 and an excess of each compound that failed to induce complex formation. A compound that binds to PPAR ⁇ without inducing complex formation would be expected to compete with Wy 14,643 thereby decreasing heterodimer formation.
- PPAR ⁇ activators such as PGA,, PGA 2 , PGB 2 , PGD 2 and 15d-J 2 may be inactive precursors that are metabolized to PPAR ⁇ ligands.
- Fig. 4B the specificity of different activator classes for each member of the PPAR family was compared.
- Naturally occurring saturated long-chain FAs C12-C16 are weak activators of PPAR ⁇ and even weaker activators of PPAR ⁇ .
- the dual function long-chain FAs e.g., 2-bromopalmitate (2Br-C16) and tetradecylthioacetic acid (TTA)
- TTA tetradecylthioacetic acid
- PUFAs are efficient activators of PPAR ⁇ and PPAR ⁇ , but display little activity on PPAR ⁇ .
- 8S-HETE was specific for PPAR ⁇
- PGA preferentially activated PPAR ⁇
- All three PPAR isoforms were responsive to 15d-J 2
- the synthetic eicosanoid cPGI selectively activated PPAR ⁇ and PPAR ⁇ .
Abstract
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EP98915218A EP0979407A1 (en) | 1997-04-29 | 1998-04-01 | Methods for identifying ligands for nuclear hormone receptors |
CA002288978A CA2288978A1 (en) | 1997-04-29 | 1998-04-01 | Methods for identifying ligands for nuclear hormone receptors |
AU69458/98A AU6945898A (en) | 1997-04-29 | 1998-04-01 | Methods for identifying ligands for nuclear hormone receptors |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19855013A1 (en) * | 1998-11-20 | 2000-07-20 | Schering Ag | Androgen and progesterone receptor binding hormone response element |
US6184256B1 (en) | 1997-04-24 | 2001-02-06 | INSTITUT NATIONAL DE LA SANTé DE LA RECHERCHE MéDICALE | Methods and compositions for use in modulating expression of matrix metalloproteinase genes |
WO2002024702A1 (en) * | 2000-09-19 | 2002-03-28 | Bristol-Myers Squibb Company | Fused heterocyclic succinimide compounds and analogs thereof, modulators of nuclear hormone receptor function |
EP1386610A1 (en) * | 2002-08-02 | 2004-02-04 | Laboratoires Goemar S.A. | Medicament containing 2-HETE or 11,12-EET |
WO2012030165A2 (en) | 2010-08-31 | 2012-03-08 | 서울대학교산학협력단 | Use of the fetal reprogramming of a ppar δ agonist |
-
1998
- 1998-04-01 WO PCT/US1998/006446 patent/WO1998049555A1/en not_active Application Discontinuation
- 1998-04-01 EP EP98915218A patent/EP0979407A1/en not_active Withdrawn
- 1998-04-01 AU AU69458/98A patent/AU6945898A/en not_active Abandoned
- 1998-04-01 CA CA002288978A patent/CA2288978A1/en not_active Abandoned
Non-Patent Citations (3)
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KLIEWER S. A., ET AL.: "DIFFERENTIAL EXPRESSION AND ACTIVATION OF A FAMILY OF MURINE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 91., 1 July 1994 (1994-07-01), US, pages 7355 - 7359., XP002914194, ISSN: 0027-8424, DOI: 10.1073/pnas.91.15.7355 * |
SCHOONJANS K., STAELS B., AUWERX J.: "THE PEROXISOME PROLIFERATOR ACTIVATED RECEPTORS (PPARS) AND THEIR EFFECTS ON LIPID METABOLISM AND ADIPOCYTE DIFFERENTIATION.", BIOCHIMICA ET BIOPHYSICA ACTA - LIPIDS AND LIPID METABOLISM., ELSEVIER SCIENCE BV. AMSTERDAM., NL, vol. 1302., 1 January 1996 (1996-01-01), NL, pages 93 - 109., XP002914195, ISSN: 0005-2760, DOI: 10.1016/0005-2760(96)00066-5 * |
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Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
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US6184256B1 (en) | 1997-04-24 | 2001-02-06 | INSTITUT NATIONAL DE LA SANTé DE LA RECHERCHE MéDICALE | Methods and compositions for use in modulating expression of matrix metalloproteinase genes |
DE19855013A1 (en) * | 1998-11-20 | 2000-07-20 | Schering Ag | Androgen and progesterone receptor binding hormone response element |
DE19855013C2 (en) * | 1998-11-20 | 2001-09-13 | Schering Ag | Androgen and progesterone receptor binding hormone response element |
US6518041B1 (en) | 1998-11-20 | 2003-02-11 | Schering Aktiengesellschaft | Hormone response element that binds an androgen and progesterone receptor |
WO2002024702A1 (en) * | 2000-09-19 | 2002-03-28 | Bristol-Myers Squibb Company | Fused heterocyclic succinimide compounds and analogs thereof, modulators of nuclear hormone receptor function |
JP2004509895A (en) * | 2000-09-19 | 2004-04-02 | ブリストル−マイヤーズ スクイブ カンパニー | Condensed heterocyclic succinimide compounds and analogs thereof which are modulators of nuclear hormone receptor functions |
CN1307181C (en) * | 2000-09-19 | 2007-03-28 | 布里斯托尔-迈尔斯斯奎布公司 | Selective androgen receptor modulators and methods for their identification, design and use |
KR100765670B1 (en) * | 2000-09-19 | 2007-10-10 | 브리스톨-마이어스스퀴브컴파니 | Fused Heterocyclic Succinimide Compounds and Analogs Thereof, Modulators of Nuclear Hormone Receptor Function |
EP1386610A1 (en) * | 2002-08-02 | 2004-02-04 | Laboratoires Goemar S.A. | Medicament containing 2-HETE or 11,12-EET |
FR2843026A1 (en) * | 2002-08-02 | 2004-02-06 | Goemar Lab Sa | NEW MEDICINE |
WO2012030165A2 (en) | 2010-08-31 | 2012-03-08 | 서울대학교산학협력단 | Use of the fetal reprogramming of a ppar δ agonist |
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