INTRACELLULAR C-TERMINAL HDL-RECEPTOR DOMAIN

Abstract: The invention relates to new peptides corresponding substantially to the intracellular C-terminal domain of a mammalian HDL-receptor, or mimetics or functionally equivalent homologues, analogues or fragments thereof, and also to cDNA encoding such peptides. Said peptides are suitable for use in medicinal products for treatment of hyperlipidemia by modulation of cholesterol, bile acid, and/or lipid metabolism of a patient, as well as for identification of drug targets for treatment of hyperlipidemia.

Description: INTRACELLULAR C-TERMINAL HDL-RECEPTOR DOMAIN Field of the invention This invention relates to new peptides derived from mammalian HDL-receptors, to cDNA encoding such peptides, and to use thereof, particularly in treatment of hyper- lipidemia. Description of background and related art Cardiovascular disease is a leading cause of death in men >45 years and women >65 years of age in Western countries. Important risk factors include smoking, hypertension, diabetes obesity and hyperlipidemia. Hyperlipidemia is one of the major risk factors for the development of coronary artery disease and arteriosclerosis. Large scale epidemiologic studies in the 1950s dem- onstrated positive correlations between increased cholesterol levels in the blood and coronary artery disease. In the 1980s large-scale lipid-reducing intervention trials confirmed that high plasma levels of cholesterol are causally related to development of coronary artery dis- ease. These studies also showed that reduction of cholesterol levels resulted in reduced progression of arterio- sclerotic lesion and even induced regression of preexisting coronary atherosclerotic lesions, and that the lipid-reducing treatment significantly lowered the risk for clinical coronary events. Treatment of hyperlipidemia is based on dietary therapy together with hypolipidemic drugs. The most potent lipid-lowering agents currently available are the 3- hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, also known as statins. The statins were originally produced for fungal metabolites, but synthetic derivatives are now available and several different statins are currently in clinical use. The statins competitively inhibit HMG-CoA reductase, an enzyme that cata- lyzes the conversion of HMG-CoA to mevalonate, a precursor for sterols, including cholesterol. Inhibition of cholesterol synthesis leads to up-regulation of LDL- receptors in the liver and increased clearance of LDL from the blood. Statins may also influence LDL production by reducing hepatic production of very low-density lipo- protein (VLDL) and by increasing VLDL remnant catabolism. The statins decrease LDL cholesterol levels in a dose- dependent manner, and with the doses commonly used reduc- tions of 20-25% are usually seen. There is evidence to suggest that statins have additional antiatherogenic effects beyond their ability to decrease LDL-cholesterol, decrease atherosclerotic plaque and induce regression of coronary lesion. Such additional effects include improvement of endothelial dysfunction and vasomotion. Modulation of the prothrombotic state, improvements in fibrinolytic activity and inhibition of smooth muscle cell proliferation has also been observed. Long-term clinical use has shown that the statins are well tolerated and safe. Rare, but serious, side effects include hepatotoxicity and myopathy progressing to rhabdomyolysis and renal failure. A recent analysis of several trials revealed that active treatment with statins resulted in 29 % reduction in the risk of stroke and 22 % reduction in total mortality, which was attributed to a 28 % reduction in death from cardiovascular disease. There is evidence that levels of high-density lipo- protein (HDL) cholesterol correlate inversely with the incidence of cardiovascular disease. It has been suggested that HDL removes excess cholesterol from blood and tissues, including atherosclerotic plaques. The HDL cholesterol is delivered to the liver and other tissues where it is used for the production of other substances, including bile acids and steroid hormones. Recent studies have demonstrated that this transport of HDL cholesterol is mediated by the HDL-receptor, SR-BI, which is ex- pressed at high levels in the liver and in steroid producing endocrine glands. The sequence of cDNA encoding the whole rat SR-BI has been determined and a putative peroxisomal targeting sequence type 1 (PTS-1) has been identified in the C- terminus of the predicted protein. Alignment of the predicted amino acid sequence of the rat SR-BI with the corresponding human, mouse and hamster sequences showed that the protein was highly conserved among species and the putative PTS-1 sequence was identical. However, the significance of this sequence has until now not been known, see e.g. J. Biol . Chem. 1998 273:15241-158248. Summary of the invention It has now been found that by using the intracellu- lar C-terminal part of the HDL-receptor it is possible to modulate the metabolism of cholesterol, bile acid and lipids and thus to treat hyperlipidemia. The present invention thus relates to new peptides corresponding substantially to the intracellular C- terminal domain of a mammalian HDL-receptor, or a mimetic or a functionally equivalent homologue, analogue or fragment thereof, as well as to corresponding cDNA. The invention also relates to medicinal products comprising such a peptide or such a cDNA, and also to use of such a peptide or such a cDNA for the production of a medicinal product for treatment of hyperlipidemia, in particular by the modulation of cholesterol, bile acid, and/or lipid metabolism of a patient. Furthermore, the invention relates to a method for treatment of hyperlipidemia, particularly by modulating the cholesterol, bile acid, and lipid metabolism of a patient . Moreover, the invention relates to use of the above mentioned peptides, or mimetics or functionally equivalent homologues, analogues or fragments thereof, for identification of drug targets for treatment of hyperlipidemia . Detailed description of the invention Mammalian HDL-receptors, such as SR-BI in rat and CLA-1 in man, consist of two intracellular parts and one extracellular part. The C-terminal intracellular part ends with a peroxisomal targeting sequence. Such motif may interact with a peroxisomal import receptor which di- rects the protein to the peroxisome. The peroxisome has recently been shown to be of great importance for the synthesis of cholesterol and bile acids. HDL-cholesterol particles have been shown to inhibit cholesterol synthesis and stimulate bile production in the liver. During the research work leading to the present invention it was found that it is possible to use peptides corresponding to this C-terminal intracellular part of a mammalian HDL- receptor in order to modulate the synthesis and secretion of cholesterol from the peroxisomes, as well as the syn- thesis and secretion of bile, and thus to treat hyperlipidemia. One possible mechanism may be that such peptides, i.e. the peptides according to the invention, alters the activity of enzymes in the peroxisome in such a way that cholesterol synthesis is inhibited and cholesterol elimination is promoted. However, the invention is not in any way bound to this mechanism. As stated above the invention relates to new peptides corresponding to the intracellular C-terminal do- main of a mammalian HDL-receptor, comprising a peroxisomal targeting sequence, or a mimetic or a functionally equivalent homologue or analogue or fragment thereof, as well as to corresponding cDNA. The term "mimetic", as it is used herein, relates to any substance or peptide structure that closely imitate a specified substance. The term "functional equivalent homologue or analogue" , as it is used herein, relates to any substance or peptide structure that resembles a specified peptide and that will have the same pharmaceutical effect as that peptide. It is also possible to use fragments of the above mentioned peptides, provided that they have substantially the same pharmaceutical effect. The term "patient", as it is used herein, relates to any human or non-human mammal in need of treatment according to the invention. The term "treatment" used herein relates to both treatment in order to cure or alleviate a disease or a condition, and to treatment in order to prevent the de- velopment of a disease or a condition. The treatment may either be performed in an acute or in a chronic way. Throughout this description, and the appended Sequence Listing and claims, the amino acids are denoted by the three-letter symbols which are known to man skilled in the art. The amino acid sequences of preferred examples of peptides according to the invention are given in the Sequence Listing, wherein sequence No. 1 is derived from rat, sequence No. 2 is derived from mouse, sequence No. 3 is derived from hamster, and sequence No. 4 is derived from man. As evident from the Sequence Listing the four sequences resemble each other closely, and the rat sequence only differs from the hamster and mouse sequence by single amino acids. It is important that the peptides according to the invention comprises, at their C-terminal end, a peroxisomal targeting sequence, such as the putative peroxisomal targeting sequence type 1, PTS-1, Ala Lys Leu or alternative sequences that utilize peroxisome import receptors. In order for the peptides according to the invention to be taken up by the cells when administer to a patient it may be necessary to alter the characteristics of the peptides, as known to man skilled in the art. The pharmaceutically effective amount of the peptide according to the invention will vary depending on the peptide used and on the desired effect. The medicinal product according to the invention may also comprise other active substances, on condition that they do not negatively interfere with intended effect of the peptide according to the invention. The medicinal product according to the invention may also comprise other substances, such as an inert vehicle, or pharmaceutically acceptable adjuvants, carriers, preservatives etc., which are well known to persons skilled in the art . The medicinal product according to the invention may be formulated in any suitable form such as tablets, cachets, capsules, granules, oral solutions, injection solutions, infusion solutions etc.; such formulation is known to persons skilled in the art. The invention also relates to cDNA corresponding to the above mentioned peptides. The sequence of a preferred example of such a cDNA is given in sequence No. 5 in the Sequence Listing. This cDNA can e.g. be used for the production of the peptides according to the invention or be used in gene therapy and related technologies targeting nucleic acids. As stated above, the invention also relates to the use of a peptide according to the invention, or a mimetic or a functionally equivalent homologue, analogue or frag- ment thereof, for the identification of drug targets for treatment of hyperlipidemia. For example, the peptide can be used for identification of molecules involved in cholesterol or lipid metabolism with which it interacts as well as for identifi- cation of regulatory domains of such molecules. Examples of technologies that may be used for such strategies include co-immuno-precipitation experiments and cloning by use of yeast two hybrid systems. Such target molecules and domains with which the peptide interacts can be used for identification and design of small molecules that mimic the effects of the peptide. The invention will be more fully understood when reading the following examples. They should not, however, be construed as limiting the scope of the invention. Brief description of the figures In the examples below reference is made to the appended drawings on which: Figure 1 shows two expression vectors for IC-SR-BI- FLAG and IC-SR-BIΔ (AKL) -FLAG; Figure 2 is a Western blot showing the expression of IC-SR-BI-FLAG and IC-SR-BIΔ (AKL) -FLAG; Figure 3A illustrates the cellular distribution of IC-SR-BI-FLAG determined by immunohistochemistry using an antibody directed against the FLAG epitope; Figure 3B illustrates the immunohistochemical iden- tification of peroxisomes using an antibody directed against the peroxisome specific enzyme catalase. Labeling was done on the same cell as in figure 3A; Figure 3C Cellular distribution of IC-SR-BIΔ (AKL) - FLAG determined by immunohistochemistry using an antibody directed against the FLAG epitope; Figure 3D illustrates the immunohistochemical identification of peroxisomes using an antibody directed against the peroxisome specific enzyme catalase. Labeling was done on the same cell as in figure 3C. Figure 4 illustrates the effect of IC-SR-BI-FLAG and IC-SR-BIΔAKL-FLAG on cholesterol synthesis compared to a control group, the results are expressed as % of mock transfected controls. Examples EXAMPLE 1 Vectors for expression in mammalian cells of the C- terminal domain of SR-BI, with (pIC-SR-BI-FLAG) and with- out (pIC-SR-BlΔ (AKL) -FLAG) the C-terminal PTS-1 motif (AKL) , fused to two copies of the FLAG epitope (Asp Tyr Lys Asp Asp Asp Asp Lys) . The first FLAG epitope was preceded by a Met and separated from the second FLAG epitope by six amino acids (Leu Ala Ala Ala Asm Ser) . The sequence containing the two FLAG epitopes was linked via a spacer (Gly Gly Gly Gly) to the C-terminal domain of SR- BI, with (pIC-SR-BI-FLAG) and without (pIC-SR-BlΔ (AKL) - FLAG) the C-terminal PTS-1 motif (Ala Lys Leu) . The con- structs were made using standard methods (see e.g. Current protocols in molecular biology, John Wiley and Sons, Inc., PA U.S.A.). Figure 1 illustrates two different expression vectors. The expression vectors were transfected into CHO- cells by use of lipofection. Two days after the transfec- tion, the expression of the fusion proteins was verified by immunoprecipitation followed by Western blot analysis. The result is shown in figure 2. An anti-FLAG antibody (Mouse anti-FLAG M2 , Sigma Chemical Co., MO, U.S.A.) was used for immunoprecipitation and detection of the fusion proteins . Thereafter the cellular localization of IC-SR-BI- FLAG and IC-SR-BIΔ (AKL) -FLAG was determined by immunohistochemistry using an antibody directed against the FLAG epitope (Mouse anti-FLAG M2) and against catalase (sheep anti-bovine catalase, Biodesign International, ME, U.S.A.), a marker for peroxisomes. CHO cells were transfected with pIC-SR-BI-FLAG and pIC-SR-BlΔ (AKL) -FLAG and subjected to double immunofluorescence staining using the anti-FLAG antibody and the anti-catalase antibody and fluorescent secondary antibodies. In this experiment Fluorescein (FITC) -conjugated Affinipure F(ab')2 Fragment Donkey Anti-Mouse IgG (H+L) (Jackson ImmunoResearch, PA, U.S.A) and Texas Red-conjugated Affinipure F(ab')2 Frag- ment Donkey Anti-Sheep IgG (H+L) (Jackson ImmunoResearch, PA, U.S.A) were used as secondary antibodies for the anti-FLAG and anti-catalase antibodies, respectively. The results were visualized by confocal microscopy, see figures 3A-3D. In contrast to IC-SR-BIΔ (AKL) -FLAG, IC-SR-BI- FLAG co-localized with catalase (a marker for peroxisomes) . EXAMPLE 2 The effect of IC-SR-BI-FLAG on cholesterol synthesis was analyzed in cells transfected with pIC-SR-BI-FLAG, pIC-SR-BlΔ (AKL) -FLAG, or control plasmid. The effect of IC-SR-BI-FLAG and IC-SR-BIΔAKL-FLAG on incorporation of [ 14 C] acetate incorporated into cholesterol was analyzed essentially as described in J. Biol . Chem. 1974 249:5153- 5162. Cells transfected with pIC-SR-BI-FLAG incorporated less [ 14 C] acetate incorporated into cholesterol compared to cells transfected with pIC-SR-BlΔ (AKL) -FLAG, or a control plasmid. The results, illustrated in figure 4, are expressed as % of mock transfected controls.

Claims: CLAIMS 1. A peptide corresponding substantially to the intracellular C-terminal domain of a mammalian HDL- receptor, or a mimetic or a functionally equivalent homo- logue, analogue or fragment thereof. 2. A peptide according to claim 1 having a sequence selected from the group consisting of sequences No. 1-4 given in the Sequence Listing. 3. A cDNA encoding the peptide according to claim 1 or claim 2. 4. A cDNA according to claim 3, the sequence of which is sequence No. 5 given in the Sequence Listing. 5. Use of a cDNA according to claim 3 or claim 4 in gene therapy. 6. A medicinal product comprising a peptide according to claim 1 or 2 , or a mimetic or a functionally equivalent homologue, analogue or fragment thereof, and optionally other active substances and/or excipients. 7. A medicinal product according to claim 6 for treatment of hyperlipidemia. 8. A medicinal product according to claim 7 for the modulation of cholesterol, bile acid, and/or lipid metabolism of a patient. 9. Use of a peptide according to claim 1 or 2 , or a mimetic or a functionally equivalent homologue, analogue or fragment thereof, for the identification of drug targets for treatment of hyperlipidemia. 10. Use according to claim 9, wherein the drug tar- gets identified are drug targets for modulation of the metabolism of cholesterol, bile acid, and/or lipids. 11. Use of a peptide corresponding substantially to the intracellular C-terminal domain of a mammalian HDL- receptor, or a mimetic or a functionally equivalent homo- logue, analogue or fragment thereof for the production of a medicinal product for treatment of hyperlipidemia. 12. Use according to claim 11, for the modulation of cholesterol, bile acid, and/or lipid metabolism of a patient . 13. Use according to claim 11 or claim 12, wherein said peptide has a sequence selected from the group consisting of sequences No. 1-4 given in the Sequence Listing. 14. A method for treatment of hyperlipidemia comprising administration to a patient a pharmaceutically active amount of a peptide corresponding substantially to the intracellular C-terminal domain of a mammalian HDL- receptor, or a mimetic or a functionally equivalent homologue, analogue or fragment thereof. 15. A method according to claim 14, wherein the treatment of hyperlipidemia is accomplished by the modulation of cholesterol, bile acid, and lipid metabolism of the patient. 16. A method according to claim 14 or claim 15, wherein said peptide has a sequence selected from the group consisting of sequences No. 1-4 given in the Sequence Listing.

Inventors: CARLSSON BJOERN, CARLSSON LENA, JOHNSON MAGNUS, CARLSSON, BJOERN, CARLSSON, LENA, JOHNSON, MAGNUS
Applicants: CARLSSON BJOERN, CARLSSON LENA, JOHNSON MAGNUS, CARLSSON, BJOERN, CARLSSON, LENA, JOHNSON, MAGNUS
Application year: 1999
Main classification: C07K14
IPC codes: A61K38, A61K48, C07K14