WO2017207733A1 - Recombinant sirt1 - Google Patents
Recombinant sirt1 Download PDFInfo
- Publication number
- WO2017207733A1 WO2017207733A1 PCT/EP2017/063395 EP2017063395W WO2017207733A1 WO 2017207733 A1 WO2017207733 A1 WO 2017207733A1 EP 2017063395 W EP2017063395 W EP 2017063395W WO 2017207733 A1 WO2017207733 A1 WO 2017207733A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sirt1
- patient
- pcsk9
- level
- isolated polypeptide
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
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- G—PHYSICS
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/50—Determining the risk of developing a disease
Definitions
- the present invention relates to the use of recombinant Sirtuin-1 (SIRT1 ) for use in the treatment of conditions associated with aging, elevated cholesterol levels and dyslipidemia, such as obesity, atherosclerosis, pancreatitis, coronary artery disease or steatohepatitis.
- SIRT1 recombinant Sirtuin-1
- SIRT1 relates to Sirtuin-1 (silent mating type information regulation 2 homologue 1 ).
- SIRT1 is a highly conserved protein deacetylase that requires NAD + (nicotinamide adenine dinucleotide) as a co-substrate.
- NAD + nicotinamide adenine dinucleotide
- the deacetylation of acetyl-lysines by Sirtuin 1 is coupled with NAD + hydrolysis, producing nicotinamide and an acetyl-ADP ribose compound.
- Sirtuin 1 also exhibits NAD + -dependent histone deacetylase activity.
- rSIRTI refers to a SIRT1 polypeptide that has been produced using biotechnological methods.
- the term refers to human SIRT1 isoform 1 (NCBI Reference Sequence NP_036370.2).
- the term refers to human SIRT1 isoform 2.
- sequence identity and percentage of sequence identity refer to the values determined by comparing two aligned sequences.
- Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981 ), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).
- sequence identity values refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.
- LDL low density lipoprotein
- lipid including cholesterol, cholesterol esters and triglycerides.
- LDL includes primarily apolipoprotein B-100 and apolipoprotein E. LDL incorporates and transports cholesterol in the plasma.
- the term LDL may be used in a context-dependent manner to designate cholesterol bound to LDL particles.
- LDLR relates to low density lipoprotein receptor.
- PCSK9 relates to proprotein convertase subtilisin/kexin type 9.
- PCSK9 is able to bind to LDLR and effects LDLR degradation. Inhibition of PCSK9 causes an increase in LDLR expression and thus reduces plasma LDL- cholesterol levels.
- acetylation and deacetylation are used in their meaning known in the art of biochemistry and cell biology; it refers to a modification of proteins, where acetyl groups are covalently attached to or removed from lysine residues within the protein. This modification is known to affect the properties and functions of the proteins.
- Acetylation of proteins is catalyzed by acetyltransferases, whereas deacetylation is catalyzed by deacetylases.
- cardiovascular disease is used to classify conditions that affect the heart, heart valves, blood, and vasculature of the body, particularly diseases affected by an aberrant plasma level of lipids, more particularly by an aberrant level of cholesterol, LDL, HDL and/or VLDL.
- Cardiovascular diseases that can be modified by the invention include endothelial dysfunction, coronary artery disease (CAD), angina pectoris, myocardial infarction, acute coronary syndrome (ACS), atherosclerosis, congestive heart failure, hypertension, cerebrovascular disease, stroke, transient ischemic attacks, deep vein thrombosis, peripheral artery disease, cardiomyopathy, arrhythmias, aortic stenosis, and aneurysm.
- CAD coronary artery disease
- ACS acute coronary syndrome
- atherosclerosis congestive heart failure
- hypertension cerebrovascular disease
- stroke stroke
- transient ischemic attacks deep vein thrombosis
- peripheral artery disease CAD
- cardiomyopathy arrhythmias
- arrhythmias aortic stenosis
- aneurysm aneurysm
- Particular diseases that are amenable to treatment according to the invention include atherosclerosis-associated endothelial dysfunction, atherosclerosis- associated coronary artery disease (CAD), atherosclerosis-associated angina pectoris, atherosclerosis-associated myocardial infarction, atherosclerosis-associated acute coronary syndrome (ACS), atherosclerosis-associated congestive heart failure, atherosclerosis- associated hypertension, atherosclerosis-associated cerebrovascular disease, stroke, atherosclerosis-associated transient ischemic attacks, atherosclerosis-associated deep vein thrombosis, atherosclerosis-associated peripheral artery disease, atherosclerosis-associated cardiomyopathy, atherosclerosis-associated arrhythmias, aortic stenosis, and atherosclerosis-associated aneurysm.
- CAD atherosclerosis-associated coronary artery disease
- ACS atherosclerosis-associated acute coronary syndrome
- atherosclerosis-associated congestive heart failure atherosclerosis- associated hypertension, atherosclerosis-associated
- diseases that are amenable to treatment according to the invention include dyslipidemia-associated endothelial dysfunction, dyslipidemia-associated coronary artery disease (CAD), dyslipidemia-associated endothelial dysfunction, dyslipidemia-associated angina pectoris, dyslipidemia-associated myocardial infarction, dyslipidemia-associated acute coronary syndrome (ACS), dyslipidemia-associated congestive heart failure, dyslipidemia-associated hypertension, dyslipidemia-associated cerebrovascular disease, stroke, dyslipidemia-associated transient ischemic attacks, dyslipidemia-associated deep vein thrombosis, dyslipidemia-associated peripheral artery disease, dyslipidemia-associated cardiomyopathy, dyslipidemia-associated arrhythmias, aortic stenosis, and dyslipidemia- associated aneurysm.
- CAD dyslipidemia-associated coronary artery disease
- ACS dyslipidemia-associated myocardial infarction
- dyslipidemia-associated acute coronary syndrome ACS
- dyslipidemia-associated congestive heart failure dyslipidemia-associated
- CAD coronary artery disease
- IHD ischemic heart disease
- CAD comprises a group of diseases including stable angina, unstable angina, myocardial infarction and sudden cardiac death.
- a particularly relevant CAD indication for treatment with the invention is ST-Elevation Myocardial Infarction (STEMI).
- ACS acute coronary syndrome
- a first aspect of the invention provides an isolated polypeptide comprising an amino acid sequence characterized by at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, 96%, >97%, >98%, >99%, >99.5%, >99.9% sequence identity to SEQ ID NO 01 or SEQ ID NO 02 for use in a method of treatment or prevention of a condition selected from the group comprising dyslipidemia, hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, diabetes, particularly diabetes type II, metabolic syndrome and obesity-related conditions.
- the isolated polypeptide has a NAD + -dependent deacetylase function identical to SIRT1 isoform 1 or isoform 2.
- polypeptide has a NAD + - dependent deacetylase function identical to SIRT1
- polypeptide that exhibits at least 85% of the deacetylase activity of SIRT1 as determined in the examples provided herewith, particularly by the method described in the experiment documented in Fig. 2 or Fig. 7, particularly as documented in Fig. 7 and the associated description of the example.
- rSIRTI protects against atherosclerosis in mice (Fig. 3), indicating that rSIRTI is also protective against atherosclerosis in humans.
- treatment with rSIRTI reduces steatohepatitis in mice (Fig. 19) and decreases the number of foam cells (inflammatory macrophages accumulating oxidized fat) in liver and kidney of mice (Fig. 8).
- rSIRTI reduces plasma glucose levels (Fig. 10 a), increases non-fasting plasma insulin levels (Fig. 10 b) and increases insulin-producing ⁇ -cells in the pancreas of mice (Fig. 10 d).
- Administration of rSIRTI is thus a promising strategy for treatment of diabetes.
- rSIRTI reduces plasma lipids/cholesterol in mice (Fig. 8, Fig. 22), indicating that rSIRTI is useful in the treatment of hyperlipidemia, dyslipidemia and hypercholesterolemia.
- Hyperlipidemia is a common cause of pancreatitis, thus reduction of blood lipids by rSIRTI treatment is protective against pancreatitis.
- Elevated plasma lipids/cholesterol are also an important risk factor underlying cardiovascular disease (CVD), therefore rSIRTI treatment is useful in the therapy or prevention of CVD.
- CVD cardiovascular disease
- a second aspect of the invention relates to a dosage form comprising the isolated polypeptide according to the first aspect of the invention for use in therapy or prevention of a condition selected from the group comprising dyslipidemia, hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, diabetes, metabolic syndrome and obesity- related conditions.
- a condition selected from the group comprising dyslipidemia, hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, diabetes, metabolic syndrome and obesity- related conditions.
- the amount of said isolated polypeptide is 1 pg to 5 mg per dose.
- the amount of said isolated polypeptide is 10 pg to 5 mg per dose.
- the amount of said isolated polypeptide is 100 pg to 3.5 mg per dose.
- the amount of said isolated polypeptide is 500 pg
- said dose is administered daily. In certain embodiments, said dose is administered twice per day. In certain embodiments, said dose is administered several times per day. In certain embodiments, said dose is administered every other day. In certain embodiments, said dose is administered weekly.
- said dosage form is formulated for parenteral, peroral, transdermal or transmucosal administration.
- a combination medicament comprising
- NAD + supplement refers to a compound selected from NAD + and precursors of NAD + .
- NAD + supplements are dietary supplements capable of rising a person's NAD + level, particularly a compound comprising a nicotinic acid or nicotinamide moiety.
- Activity of SIRT1 therapy can be increased by dual treatment with natural SIRT1 activators or NAD + supplements.
- the components of the combination medicament can be comprised in a single dosage form or in separate dosage forms.
- the components of the combination medicament may be administered simultaneously or separately.
- the SIRT1 activator is a natural SIRT1 activator. In certain embodiments of this aspect of the invention, the SIRT1 activator is a synthetic SI RT1 activator.
- SIRT1 activator refers to a compound capable of increasing the effect of SIRT1 .
- SIRT1 activators are piceatannol (CAS No. 10083-24-6), resveratrol (CAS No. 501-36-0), butein (CAS No. 487- 52-5), quercetin dihydrate (CAS No. 6151 -25-3), SIRT1 Activator 3 (CAS No. 839699-72-8), SRT1720 (CAS No. 1001645-58-4), BML-278 (CAS No. 15301-69-6).
- the combination medicament comprises the isolated polypeptide according to the first aspect of the invention and a natural or synthetic SIRT1 activator, or a natural or synthetic NAD + supplement, selected from niacin (vitamin B3), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR) and resveratrol.
- a natural or synthetic SIRT1 activator selected from niacin (vitamin B3), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR) and resveratrol.
- the combination medicament is provided for use in therapy or prevention of a condition selected from the group comprising dyslipidemia, hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, diabetes, metabolic syndrome and obesity-related conditions.
- a method of determining the risk of a patient to develop CAD comprises quantifying the level of plasma SIRT1 in a sample obtained from the patient, and using the obtained information as an intermediate result during assessment of the risk of the patient to develop CAD. It is indicative of a high risk to develop CAD if the SIRT1 level is below a predetermined threshold.
- the inventors have shown that plasma SIRT1 levels decrease with age (Fig. 4). This age- dependent decrease inversely correlates with plasma PCSK9 levels and thus with reduced LDL-cholesterol.
- the inventors further show that rSIRTI reduces plasma lipids in mice (Fig. 8, Fig. 19). Together, this indicates that plasma SIRT1 levels above a certain threshold are protective against CAD and plasma SIRT1 levels below a certain threshold are indicative of an increased risk to develop CAD.
- a method of determining the risk of a patient to develop CAD comprises quantifying the level of SIRT1 expression in a sample obtained from the patient and assigning to the patient a high risk of developing CAD if the SIRT1 level is below a predetermined threshold.
- a method of determining ACS in a patient comprises quantifying the level of SIRT1 expression in a sample obtained from the patient, and using the obtained information as an intermediate result during assessment of the probability of ACS. It is indicative of a high probability of ACS if the SIRT1 level is below a predetermined threshold.
- a method of determining ACS in a patient comprises quantifying the level of SIRT1 expression in a sample obtained from the patient and assigning to the patient a high probability of ACS if the SIRT1 level is below a predetermined threshold.
- said methods comprise contacting the sample obtained from the patient with an antibody against SIRT1.
- said quantification of SIRT1 expression levels is done by ELISA.
- an antibody against SIRT1 in the preparation of a kit for determining the risk of a patient to develop CAD is provided.
- an antibody against SIRT1 in the preparation of a kit for diagnosing ACS is provided.
- a method of treatment of a condition selected from the group comprising dyslipidemia, hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, diabetes, metabolic syndrome and obesity-related conditions comprises administration of an isolated polypeptide comprising an amino acid sequence characterized by at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, 96%, >97%, >98%, >99% sequence identity to SEQ ID NO 01 or SEQ ID NO 02 to a patient in need thereof.
- the method comprises improving lipid profiles towards normal levels, in other words towards levels found in a person not diagnosed with any of the above mentioned diseases.
- the method comprises improving HDL functionality.
- the method comprises improving glucose sensitivity and insulin sensitivity.
- the method comprises reducing body weight in patients diagnosed with obesity.
- Fig. 1 shows that SIRT1 isoforms function differentially in Huh7 cells.
- Veh vehicle; rSIRTI , recombinant SIRT1 iso-1 ; 3KR, deacetyl mimetic PCSK9 with mutations at K243R/K421 R/K506R; 3KQ, acetyl mimetic PCSK9 with mutations at K243Q/K421 Q/K506Q; EGF-AB, epidermal growth factor-AB; AU, arbitrary units.
- Fig. 3 shows that recombinant SIRT1 treatment protects against atherosclerosis and reduces plasma LDL-cholesterol in ApoE-/- mice.
- Fig. 4 shows a correlation of plasma SIRT1 and human pathophysiology
- (a) Bar graph of an ELISA of plasma SIRT1 levels in healthy subjects of different age groups. Each bar represents 10 years beginning with 21 years of age with an n 20 per decade
- Fig. 5 shows that SIRT1 is secreted in plasma and modulates gene expression
- (a) Relative gene expression of genes involved in glucose and lipid metabolism after treatment with rSIRTI for 8h in the presence of serum (24h) (n 8 per group). Gene expression is normalized to Rps29.
- Fig. 6 shows that SIRT1 modulates LDLR expression and PCSK9 activity
- Fig. 7 shows identification and mapping of PCSK9 (SEQ ID NO 033) acetylation and deacetylation by SIRT1 .
- Recombinant PCSK9 was incubated with recombinant SIRT1 with and without NAD+.
- the protein from the Colloidal Coomassie stained band was digested with trypsin and the resulting peptides were analyzed by LC-MS/MS and MALDI MS/MS.
- MS/MS data were analyzed using the sequences of PCSK9 with the Mascot algorithm, allowing the detection of acetylated lysine residues,
- c MS/MS spectrum of all peptides recovered in the LC-MS/MS experiment for PCSK9 after performing PCSK9 deacetylation assay, highlighted in yellow and lysine modification highlighted by green Acetylation was detected lysine residue 243, 273, 421 and 506 in the fragmentation spectrums of peptides from trypsin- digested PCSK9.
- Fig. 8 shows that Recombinant SIRT1 treatment reduces macrophage accumulation reduces and plasma VLDL-cholesterol in ApoE-/- mice.
- Representative pictures immunohistochemically stained with H&E.
- (c, d) Bar graph of plasma VLDL cholesterol and HDL-cholesterol concentrations.
- Veh vehicle
- rSIRTI recombinant SIRT1 iso-1 ;
- AU arbitrary units;
- VLDL very low- density lipoprotein; HDL, high-density lipoprotein.
- Fig. 9 shows the effect of recombinant SIRT1 treatment in ApoE-/- mice.
- Veh vehicle; rSIRTI , recombinant SIRT1 iso-1 ; epiWAT, epididymal white adipose tissue.
- Fig. 10 shows the effect of recombinant SIRT1 treatment on glucose metabolism in ApoE-/- mice.
- Fig. 1 1 shows the correlation of plasma SIRT1 and and PCSK9 in healthy subjects
- Fig. 13 shows that rSIRTI treatment improves glucose sensitivity in wild-type mice on high- fat diet.
- Fig. 15 shows that rSIRTI treatment increases liver weight in ApoE " ' " mice.
- Fig. 16 shows that rSIRTI treatment tends to reduce steatohepatitis in ApoE " ' " mice.
- Fig. 17 shows that SIRT1 circulates in the plasma. Representative western blot of SIRT1 immunoprecipitated from plasma of healthy subjects.
- Fig. 18 shows the time-course of SIRT1 treatment (2 pg) in Huh7 cells. Representative western blot of SIRT1 , LDLR, PCSK9 and ⁇ -actin of Huh7 cells treated with recombinant SIRT1 .
- Fig. 19 shows that rSIRTI treatment reduces plasma lipids in ApoE " ' " mice.
- Fig. 20 shows that rSIRTI treatment does not alter plasma PCSK9 levels of ApoE " ' " mice.
- SIRT1 is a member of the Sirtuin family of NAD + - dependent deacetylase enzymes and an important regulator of nutrient metabolism and aging. Recently, several studies have attempted to detect SIRT1 in the plasma of different patient cohorts. Thus the inventors decided to investigate whether SIRT1 is secreted by hepatocytes and if so, to which of the two isoforms of SIRT1 this phenomenon be attributed to. To this end, the inventors performed isoform-specific overexpression of GFP-tagged SIRT1 in Huh7 cells and analysed the secretion of SIRT1 using a GFP ELISA (Fig. 1 a).
- SIRT1 isoform 1 (iso-1 ) was secreted to a substantially higher degree suggesting that iso-1 is the dominant secreted isoform.
- SIRT1 is involved in different metabolic pathways, the inventors determined, if isoform- specific SIRT1 differentially regulates intracellular metabolic pathways. The inventors found five genes to be differentially regulated by the two isoforms. A significantly higher expression of SLC2A4 and FASN was observed in SIRT1 iso-2 overexpressing Huh7 cells compared to empty vector and SIRT1 iso-1 (Fig. 1 b). Correspondingly, SIRT1 iso-1 decreased the expression of HK2 and PCK1 .
- SIRT1 iso-1 recombinant SIRT1 iso-1
- Huh7 cells Huh7 cells
- rSIRTI recombinant SIRT1 iso-1
- rSIRTI increased FABP4 suggesting that secreted SIRT1 alone has a different but significant in metabolic homeostasis compared to intracellular SIRT1 .
- Intracellular SIRT1 is induced upon nutrient deprivation, while not being transcriptionally increased.
- rSIRTI increased LDLR protein expression in a time-dependent manner with a maximal effect at 8 h after addition (Fig. 6a). At this point it could not be excluded that the gradual rise in LDLR expression is due to the fact that rSIRTI has used NAD + produced by the cell (as NAD + was not added externally). The inventors further observed a time-dependent manner increase in SIRT1 expression, suggesting an uptake of SIRT1 .
- LDLR expression is regulated post-transcriptionally by plasma PCSK9.
- rSIRTI did not affect gene expression of LDLR or PCSK9 at 8 h compared to vehicle (Fig. 6b), thus prompting us to examine if SIRT1 iso-1 directly binds to PCSK9 while in circulation. Pulldown experiments from normolipidemic human plasma revealed that SIRT1 was indeed bound to PCSK9, thus implying that SIRT1 may directly deacetylate PCSK9 in plasma (Fig. 2a).
- rPCSK9 recombinant PCSK9
- rSIRTI were incubated with or without co-factor NAD + in vitro and analysed by Western blot analysis. In samples incubated with NAD + , levels of acetyl-lysine were reduced compared to those without NAD + (Fig. 2b), further confirming that PCSK9 is a substrate of SIRT1 .
- SIRT1 iso-1 is a modulator of PCSK9, is bound to HDL in plasma and that LDLR is a receptor of plasma SIRT1 iso-1 .
- circulating SIRT1 iso-1 is a novel regulator of hepatic cholesterol metabolism.
- Aortic root lesions of rSIRTI -treated ApoE " ' " mice showed a significant reduction in plaque area along with macrophage accumulation (Fig. 3b and 3c) compared with vehicle-treated ApoE " ' " mice.
- the inventors found an overall decrease in foam cell formation in the liver and kidneys of rSIRTI -treated mice compared to vehicle- treated control mice (Fig. 8a, b).
- SIRT1 iso-1 modulates PCSK9
- rSIRTI -treated group they found a marked decrease in plasma total-cholesterol, LDL-C and VLDL-C (Fig. 3e, 3f, Fig. 8c), with no change in HDL- C (Fig. 8d) compared to vehicle-treated controls.
- Deficiency for Sirtl in wild-type mice has previously been shown to reduce body weight while activation of Sirtl in ApoE " ' " mice reduces body weight.
- the inventors observed no difference in body weight, food intake or epididymal white adipose tissue weight between the two groups (Fig. 9a-c). Since SIRT1 modulates glucose metabolism and improves insulin sensitivity, the inventors measured plasma glucose levels which in the non-fasting state were significantly lower in rSIRTI - treated ApoE " ' " mice (Fig. 10a), and a significant increase in plasma insulin levels (Fig. 10b).
- PCSK9 deficiency in pancreatic islets of mice caused a two-fold increase in LDLR protein expression in pancreatic islets, mainly beta cells.
- the inventors quantified ⁇ -cell and a-cell numbers using insulin as a surrogate marker for ⁇ -cells in islets and glucagon as a surrogate marker for a-cells in islets, respectively. They found a significant increase in the number of ⁇ -cells, with no difference in a-cells between the two groups (Fig. 10d, e). Thus, rSIRTI exhibits an increase in ⁇ -cell islet biogenesis.
- SIRT1 protects against atherosclerosis by decreasing plasma LDL-C and by improving glucose metabolism.
- the inventors measured plasma SIRT1 levels in healthy subjects of different age groups. Importantly, they found an age-dependent decrease in circulating SIRT1 in healthy subjects (Fig. 4a) which inversely correlated to plasma LDL-C (Fig. 4b). Furthermore, SIRT1 significantly correlated with non-HDL cholesterol and ApoBI OO plasma levels (Table 1 ). Consequently, the inventors found a significant inverse correlation between plasma SIRT1 and PCSK9 levels (Fig. 1 1 a), implicating that circulating SIRT1 is an age-dependent marker of lipid homeostasis and that the age-dependent decrease in SIRT1 contributes to the age-dependent increase in plasma LDL-C and risk to cardiovascular disease.
- the inventors extended their findings further to dyslipidemia, i.e. in a cohort of non-statin treated obese, but non-diabetic individuals. They found a significant inverse correlation between circulating SIRT1 and plasma PCSK9 and LDL-C levels. Myocardial infarction is the final result of high LDL and in turn plaque formation. Of note, in an acute setting of infarction, plasma LDL-C decline rapidly within 24h post-MI. Recently high levels of PCSK9 have been associated with an increase in inflammation in the acute phase of myocardial infarction in a large Swiss multicentre cohort study.
- the inventors here provide the first evidence that SIRT1 is secreted and that circulating SIRT1 regulates plasma PCSK9 activity by deacetylating specific acetylation sites on the protein and in turn reduces LDL-C and atherosclerotic plaque size and composition. Furthermore, the inventors show that SIRT1 is bound to HDL in circulation, binds to hepatic LDLRs and thereby cleared from circulation. Finally, recombinant SIRT1 provides a novel cardiovascular treatment modality to reduce plaque size and composition and to improve glucose homeostasis.
- Huh7, human hepatoma cells were cultured in DMEM (Sigma) supplemented with Glutamax. Where indicated cells were treatment with PBS (vehicle) or recombinant SIRT1 (500 ng) for a period of 6 hours or 1 hour. Where indicated cells were treated with cycloheximide, Brefeldin A or Methylamine for a period of 1 hour. LDH assay for toxicity was performed using manufacturers protocol. Recombinant active PCSK9 (Cusabio, Abeam) for cell culture were analysed by mass spectrometry and used for uptake analysis at indicated concentrations.
- Total cholesterol plasma concentration is measured using a colorimetric method (BioMerieux, France). Plasma sample is loaded on a gel chromatography column, which separates lipoproteins according to their size. The chromatograph effluent is immediately and continuously mixed with cholesterol assay reactant, and incubated at the appropriate conditions of temperature and duration to allow the enzymatic reaction to occur. The OD measured at the appropriate wavelength is automatically converted in a graphic signal representing the lipid distribution profile. The area under each peak is proportional to the lipid concentration in the respective lipoprotein fraction.
- PCSK9-LDLR interaction was assessed using a CircuLex PCSK9-LDLR in vitro binding assay kit (MBL International, Woburn, MA, USA) with minor modifications.
- Recombinant His- tagged PCSK9 (1 pg/mL) or recombinant PCSK9 at a concentration of 100 pg/mL were subject to a deacetylation assay for 1 h at room temperature with gentle shaking.
- the mixtures were added to an ELISA plate that was coated with EGF-AB peptide of the LDLR. Subsequent procedures were performed according to the manufacturer's instructions. Where required the detection antibody was replaced with his-tag antibody and anti-PCSK9 antibody.
- Western blotting Western blotting
- Protein from tissues and cells was prepared as described and analysed using standard protocols.10 Protein samples were immunostained using specific antibodies targeted to SIRT1 (Cell signalling), LDL-R (Novus Biologicals), PCSK9 (Abeam) or ⁇ -actin, and detected by horseradish peroxidase chemiluminescence.
- SIRT1 Cell signalling
- LDL-R Novus Biologicals
- PCSK9 Abeam
- ⁇ -actin horseradish peroxidase chemiluminescence.
- SIRT1 isoform 1 EX-U1443-M29, Genecopoeia
- SIRT1 isoform 2 EX-Z6705-M29, Genecopoeia
- wild-type PCSK9 RC220000, Origene
- Recombinant PCSK9 was incubated with recombinant SIRT1 in a buffer (250mM Tris-HCI, 20mM MgCI2, 250mM NaCI) in the presence and absence of 5mM NAD + for one hour at 37°C. The reaction was stopped using laemmeli buffer and subject to western blotting.
- a buffer 250mM Tris-HCI, 20mM MgCI2, 250mM NaCI
- Huh7 cells were cultured in a six-well plate and incubated with recombinant SIRT1 at 500ng concentration for 1 h. After treatment, cells were washed twice with phosphate-buffered saline and then incubated with 5 pg/ml of Bodipy-labelled human LDL (L-3483, Invitrogen) in serum-free medium for 1 h at 37 °C. After washing twice with phosphate-buffered saline, uptake of Bodipy-LDL-C was determined by flow cytometry.
- PCSK9 antibody (RnD Systems) was covalently linked to beaded agarose resin via primary amines using the Aminolink Plus immobilization kit (Pierce, catalog number 44894). Serum samples (200 ⁇ ) were then diluted 5-fold in immunoprecipitation buffer [50 mM HEPES (pH 7.40), 150 mM NaCI, 1 % Triton X-100, 5 mM EDTA, and 5 mM EGTA] for a total of 1 ml volume and incubated overnight with 20 pg of PCSK9 antibody linked to 40 ⁇ packed beads. The beads were washed five times with PBS + 0.1 % NP-40 used for Western blotting analysis.
- ELISA ELISA
- ELISA for mouse PCSK9 in the plasma was performed using Mouse ELISA kit (RnD Systems).
- ELISA for GFP (Abeam), human SIRT1 (Cusabio) and PCSK9 (RnD Systems) was performed using manufacturers protocol with appropriate dilutions (no dilution for cell culture supernantant, 1 :2 for SIRT1 and 1 :20 for PCSK9). All ELISA analysis was performed in samples without freeze-thaw.
- Excised gel bands were cut into approximately 1 mm 3 pieces. Gel pieces were washed twice with 100 mM ammonium bicarbonate/50% acetonitrile for 15 min at 50°C and dehydrated with acetonitrile for 10 min. All supernatants were discarded. Rehydration of the gel pieces was with 10 mM Tris-HCI, 2 mM CaCI 2 , pH 8.2 containing 5 ng/ ⁇ proteomics-grade recombinant trypsin (Roche, Diagnostics, Mannheim, Germany) at 4°C. Microwave assisted digestion (Model Discover, CEM, Matthews, NC) was performed for 30 min at 5 W and 60 C.
- Full scan MS spectra were acquired in positive profile mode from 350 - 1500 m/z with an automatic gain control target of 3e6, an Orbitrap resolution of 70 ⁇ 00 (at 200 m/z), and a maximum injection time of 100 ms.
- the 12 most intense multiply charged (z ⁇ +2) precursor ions from each full scan were selected for higher-energy collisional dissociation fragmentation with a normalized collision energy of 25 (arbitrary unit).
- Generated fragment ions were scanned with an Orbitrap resolution of 35 ⁇ 00 (at 200 m/z) an automatic gain control value of 1 e5 and a maximum injection time of 120 ms.
- the isolation window for precursor ions was set to 2.0 m/z and the underfill ratio was at 3.5% (refereeing to an intensity threshold of 2.9e4).
- Each fragmented precursor ion was set onto the dynamic exclusion list for 40 s.
- Peptides were identified by aligning the Maldi-TOF-TOF data with the known sequence of PCSK9 using the BioTools program (Bruker, Bremen, Germany) or by searching the SwissProt database (version 2015_1 1 , 549832 entries) using the Mascot search engine (Matrix Science, version 2.4.1 ). Mascot was set up to search the SwissProt database assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.030 Da and a parent ion tolerance of 10.0 PPM. Oxidation of methionine and acetylation of lysine was specified in Mascot as a variable modification.
- Scaffold version Scaffold_4.4.8, Proteome Software Inc. was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they achieved an FDR less than 0.1 % by the Scaffold Local FDR algorithm. Protein identifications were accepted if they achieved an FDR less than 1.0% and contained at least 2 identified peptides.
- SEQ ID NO 003 to SEQ ID NO 032 are listed in Table 2.
Abstract
The present invention relates to the use of an isolated polypeptide comprising an amino acid sequence characterized by at least (≥)85%, ≥87.5%, ≥90%, ≥92%, ≥94%, ≥95%, 96%, ≥97%, ≥98%, ≥99% sequence identity to SEQ ID NO 01 or SEQ ID 02. The isolated polypeptide has a NAD+-dependent deacetylase function identical to SIRT1 and is provided 5 for use in a method of treatment or prevention of dyslipidemia, hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, diabetes, metabolic syndrome and obesity-related conditions. The invention further relates to a method of determining acute coronary syndrome or determining the risk of a patient to develop coronary artery disease, comprising 10 the quantification of SIRT1 expression levels.
Description
Recombinant SIRT1
The present invention relates to the use of recombinant Sirtuin-1 (SIRT1 ) for use in the treatment of conditions associated with aging, elevated cholesterol levels and dyslipidemia, such as obesity, atherosclerosis, pancreatitis, coronary artery disease or steatohepatitis. Description of the invention
Terms and definitions
Within the context of the present specification, "SIRT1 " relates to Sirtuin-1 (silent mating type information regulation 2 homologue 1 ). SIRT1 is a highly conserved protein deacetylase that requires NAD+ (nicotinamide adenine dinucleotide) as a co-substrate. The deacetylation of acetyl-lysines by Sirtuin 1 is coupled with NAD+ hydrolysis, producing nicotinamide and an acetyl-ADP ribose compound. Sirtuin 1 also exhibits NAD+-dependent histone deacetylase activity.
Within the context of the present specification, "recombinant SIRT1 " or "rSIRTI " refers to a SIRT1 polypeptide that has been produced using biotechnological methods. In certain embodiments, the term refers to human SIRT1 isoform 1 (NCBI Reference Sequence NP_036370.2). In certain embodiments, the term refers to human SIRT1 isoform 2.
In the context of the present specifications the terms sequence identity and percentage of sequence identity refer to the values determined by comparing two aligned sequences. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981 ), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).
One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 1 1 , Extension 1 ; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1 .- 2; Gap costs: Linear. Unless otherwise stated, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol.
215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.
Within the context of the present specification, the term low density lipoprotein (LDL) relates to a class of plasma lipoproteins with a high proportion of lipid, including cholesterol, cholesterol esters and triglycerides. LDL includes primarily apolipoprotein B-100 and apolipoprotein E. LDL incorporates and transports cholesterol in the plasma. The term LDL may be used in a context-dependent manner to designate cholesterol bound to LDL particles.
Within the context of the present specification, LDLR relates to low density lipoprotein receptor.
Within the context of the present specification, PCSK9 relates to proprotein convertase subtilisin/kexin type 9. PCSK9 is able to bind to LDLR and effects LDLR degradation. Inhibition of PCSK9 causes an increase in LDLR expression and thus reduces plasma LDL- cholesterol levels.
In the context of the present specification the terms acetylation and deacetylation are used in their meaning known in the art of biochemistry and cell biology; it refers to a modification of proteins, where acetyl groups are covalently attached to or removed from lysine residues within the protein. This modification is known to affect the properties and functions of the proteins. Acetylation of proteins is catalyzed by acetyltransferases, whereas deacetylation is catalyzed by deacetylases.
Within the context of the present specification, the term cardiovascular disease (CVD) is used to classify conditions that affect the heart, heart valves, blood, and vasculature of the body, particularly diseases affected by an aberrant plasma level of lipids, more particularly by an aberrant level of cholesterol, LDL, HDL and/or VLDL. Cardiovascular diseases that can be modified by the invention include endothelial dysfunction, coronary artery disease (CAD), angina pectoris, myocardial infarction, acute coronary syndrome (ACS), atherosclerosis, congestive heart failure, hypertension, cerebrovascular disease, stroke, transient ischemic attacks, deep vein thrombosis, peripheral artery disease, cardiomyopathy, arrhythmias, aortic stenosis, and aneurysm. Particular diseases that are amenable to treatment according to the invention include atherosclerosis-associated endothelial dysfunction, atherosclerosis- associated coronary artery disease (CAD), atherosclerosis-associated angina pectoris, atherosclerosis-associated myocardial infarction, atherosclerosis-associated acute coronary syndrome (ACS), atherosclerosis-associated congestive heart failure, atherosclerosis- associated hypertension, atherosclerosis-associated cerebrovascular disease, stroke, atherosclerosis-associated transient ischemic attacks, atherosclerosis-associated deep vein thrombosis, atherosclerosis-associated peripheral artery disease, atherosclerosis-associated
cardiomyopathy, atherosclerosis-associated arrhythmias, aortic stenosis, and atherosclerosis-associated aneurysm.
More particularly, diseases that are amenable to treatment according to the invention include dyslipidemia-associated endothelial dysfunction, dyslipidemia-associated coronary artery disease (CAD), dyslipidemia-associated endothelial dysfunction, dyslipidemia-associated angina pectoris, dyslipidemia-associated myocardial infarction, dyslipidemia-associated acute coronary syndrome (ACS), dyslipidemia-associated congestive heart failure, dyslipidemia-associated hypertension, dyslipidemia-associated cerebrovascular disease, stroke, dyslipidemia-associated transient ischemic attacks, dyslipidemia-associated deep vein thrombosis, dyslipidemia-associated peripheral artery disease, dyslipidemia-associated cardiomyopathy, dyslipidemia-associated arrhythmias, aortic stenosis, and dyslipidemia- associated aneurysm.
Within the context of the present specification, the term coronary artery disease (CAD) is used synonymously with the term ischemic heart disease (IHD) and refers to conditions that are caused by decreased blood flow in the coronary arteries. CAD comprises a group of diseases including stable angina, unstable angina, myocardial infarction and sudden cardiac death. A particularly relevant CAD indication for treatment with the invention is ST-Elevation Myocardial Infarction (STEMI).
Within the context of the present specification, the term acute coronary syndrome (ACS) is used to describe an acute condition that usually occurs as a result of myocardial infarction or unstable angina.
Detailed description of the invention
A first aspect of the invention provides an isolated polypeptide comprising an amino acid sequence characterized by at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, 96%, >97%, >98%, >99%, >99.5%, >99.9% sequence identity to SEQ ID NO 01 or SEQ ID NO 02 for use in a method of treatment or prevention of a condition selected from the group comprising dyslipidemia, hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, diabetes, particularly diabetes type II, metabolic syndrome and obesity-related conditions. The isolated polypeptide has a NAD+-dependent deacetylase function identical to SIRT1 isoform 1 or isoform 2.
Within the context of the present specification, the expression "polypeptide has a NAD+- dependent deacetylase function identical to SIRT1 " refers to a polypeptide that exhibits at least 85% of the deacetylase activity of SIRT1 as determined in the examples provided
herewith, particularly by the method described in the experiment documented in Fig. 2 or Fig. 7, particularly as documented in Fig. 7 and the associated description of the example.
The inventors have shown that treatment with rSIRTI protects against atherosclerosis in mice (Fig. 3), indicating that rSIRTI is also protective against atherosclerosis in humans. The inventors have shown that treatment with rSIRTI reduces steatohepatitis in mice (Fig. 19) and decreases the number of foam cells (inflammatory macrophages accumulating oxidized fat) in liver and kidney of mice (Fig. 8). These findings indicate that rSIRTI treatment can counteract renal lipid deposition in humans and is beneficial for patients suffering from steatohepatitis and renal inflammation.
The inventors have shown that rSIRTI reduces plasma glucose levels (Fig. 10 a), increases non-fasting plasma insulin levels (Fig. 10 b) and increases insulin-producing β-cells in the pancreas of mice (Fig. 10 d). Administration of rSIRTI is thus a promising strategy for treatment of diabetes.
In addition, the inventors have shown that treatment with recombinant Sirtl improves insulin sensitivity and glucose sensitivity (Fig. 12, 13) and reduces body weight (Fig. 14) in wild-type mice on high-fat diet (45% kcal).
The inventors have shown that rSIRTI reduces plasma lipids/cholesterol in mice (Fig. 8, Fig. 22), indicating that rSIRTI is useful in the treatment of hyperlipidemia, dyslipidemia and hypercholesterolemia. Hyperlipidemia is a common cause of pancreatitis, thus reduction of blood lipids by rSIRTI treatment is protective against pancreatitis. Elevated plasma lipids/cholesterol are also an important risk factor underlying cardiovascular disease (CVD), therefore rSIRTI treatment is useful in the therapy or prevention of CVD.
A second aspect of the invention relates to a dosage form comprising the isolated polypeptide according to the first aspect of the invention for use in therapy or prevention of a condition selected from the group comprising dyslipidemia, hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, diabetes, metabolic syndrome and obesity- related conditions. In certain embodiments, the amount of said isolated polypeptide is 1 pg to 5 mg per dose. In certain embodiments, the amount of said isolated polypeptide is 10 pg to 5 mg per dose. In certain embodiments, the amount of said isolated polypeptide is 100 pg to 3.5 mg per dose. In certain embodiments, the amount of said isolated polypeptide is 500 pg to 3.5 mg per dose.
In certain embodiments, said dose is administered daily. In certain embodiments, said dose is administered twice per day. In certain embodiments, said dose is administered several
times per day. In certain embodiments, said dose is administered every other day. In certain embodiments, said dose is administered weekly.
In certain embodiments, said dosage form is formulated for parenteral, peroral, transdermal or transmucosal administration.
According to another aspect of the invention, a combination medicament is provided, comprising
a. the isolated polypeptide according to the first aspect of the invention and
b. a SIRT1 activator or NAD+ supplement.
In the context of the present specification, the term "NAD+ supplement" refers to a compound selected from NAD+ and precursors of NAD+. NAD+ supplements are dietary supplements capable of rising a person's NAD+ level, particularly a compound comprising a nicotinic acid or nicotinamide moiety. Activity of SIRT1 therapy can be increased by dual treatment with natural SIRT1 activators or NAD+ supplements.
The components of the combination medicament can be comprised in a single dosage form or in separate dosage forms. The components of the combination medicament may be administered simultaneously or separately.
In certain embodiments of this aspect of the invention, the SIRT1 activator is a natural SIRT1 activator. In certain embodiments of this aspect of the invention, the SIRT1 activator is a synthetic SI RT1 activator.
In the context of the present specification, the term SIRT1 activator refers to a compound capable of increasing the effect of SIRT1 . Non-limiting examples of SIRT1 activators are piceatannol (CAS No. 10083-24-6), resveratrol (CAS No. 501-36-0), butein (CAS No. 487- 52-5), quercetin dihydrate (CAS No. 6151 -25-3), SIRT1 Activator 3 (CAS No. 839699-72-8), SRT1720 (CAS No. 1001645-58-4), BML-278 (CAS No. 15301-69-6).
In certain embodiments of this aspect of the invention, the combination medicament comprises the isolated polypeptide according to the first aspect of the invention and a natural or synthetic SIRT1 activator, or a natural or synthetic NAD+ supplement, selected from niacin (vitamin B3), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR) and resveratrol.
In certain embodiments of this aspect of the invention, the combination medicament is provided for use in therapy or prevention of a condition selected from the group comprising dyslipidemia, hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, diabetes, metabolic syndrome and obesity-related conditions.
According to another aspect of the invention, a method of determining the risk of a patient to develop CAD is provided. The method comprises quantifying the level of plasma SIRT1 in a sample obtained from the patient, and using the obtained information as an intermediate result during assessment of the risk of the patient to develop CAD. It is indicative of a high risk to develop CAD if the SIRT1 level is below a predetermined threshold.
The inventors have shown that plasma SIRT1 levels decrease with age (Fig. 4). This age- dependent decrease inversely correlates with plasma PCSK9 levels and thus with reduced LDL-cholesterol. The inventors further show that rSIRTI reduces plasma lipids in mice (Fig. 8, Fig. 19). Together, this indicates that plasma SIRT1 levels above a certain threshold are protective against CAD and plasma SIRT1 levels below a certain threshold are indicative of an increased risk to develop CAD.
According to alternative aspect of the invention, a method of determining the risk of a patient to develop CAD is provided. The method comprises quantifying the level of SIRT1 expression in a sample obtained from the patient and assigning to the patient a high risk of developing CAD if the SIRT1 level is below a predetermined threshold.
According to yet another aspect of the invention, a method of determining ACS in a patient is provided. The method comprises quantifying the level of SIRT1 expression in a sample obtained from the patient, and using the obtained information as an intermediate result during assessment of the probability of ACS. It is indicative of a high probability of ACS if the SIRT1 level is below a predetermined threshold. According to alternative aspect of the invention, a method of determining ACS in a patient is provided. The method comprises quantifying the level of SIRT1 expression in a sample obtained from the patient and assigning to the patient a high probability of ACS if the SIRT1 level is below a predetermined threshold.
In certain embodiments, said methods comprise contacting the sample obtained from the patient with an antibody against SIRT1.
In certain embodiments, said quantification of SIRT1 expression levels is done by ELISA.
According to another aspect of the invention, the use of an antibody against SIRT1 in the preparation of a kit for determining the risk of a patient to develop CAD is provided.
According to yet another aspect of the invention, the use of an antibody against SIRT1 in the preparation of a kit for diagnosing ACS is provided.
According to another aspect of the invention, a method of treatment of a condition selected from the group comprising dyslipidemia, hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, diabetes, metabolic syndrome and obesity-related
conditions is provided. The method comprises administration of an isolated polypeptide comprising an amino acid sequence characterized by at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, 96%, >97%, >98%, >99% sequence identity to SEQ ID NO 01 or SEQ ID NO 02 to a patient in need thereof.
In certain embodiments of this aspect of the invention, the method comprises improving lipid profiles towards normal levels, in other words towards levels found in a person not diagnosed with any of the above mentioned diseases.
In certain embodiments of this aspect of the invention, the method comprises improving HDL functionality.
In certain embodiments of this aspect of the invention, the method comprises improving glucose sensitivity and insulin sensitivity.
In certain embodiments of this aspect of the invention, the method comprises reducing body weight in patients diagnosed with obesity.
Wherever alternatives for single separable features are laid out herein as "embodiments", it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.
The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.
Brief description of the figures
Fig. 1 shows that SIRT1 isoforms function differentially in Huh7 cells. Representative bar graph of ELISA of (a) intracellular and extracellular GFP expression from GFP-tagged SIRT1 iso-1 or iso-2 secreted from transfected Huh7 hepatoma cells compared to empty vector (E.V.) controls (n=8 per group), (b) Relative gene expression of genes involved in glucose and lipid metabolism confirm differential function of SIRT1 iso-1 or SIRT1 iso-2 overexpression in Huh7 cells compared to empty vector control (n=8 per group). Gene expression is normalized to Rps29. (c) Relative gene expression of genes involved in glucose and lipid metabolism after treatment with rSIRTI for 8h in the absence of serum (24h) (n=8 per group). Gene expression is normalized to Rps29. (d) Representative bar graph of ELISA of extracellular GFP expression from of Huh7 hepatoma cells transfected with GFP-tagged SIRT1 isoforms released upon starvation at decreasing percent of serum (n=8 per group). Data normalized to GFP levels in 10% serum. Veh, vehicle; rSIRTI , recombinant SIRT1 iso-1 ; AU, arbitrary units.
Fig. 2 shows that SIRT1 binds and deacetylates PCSK9. (a) Representative western blot of PCSK9 and SIRT1 expression in Huh7 cells as control and co-immunoprecipitated PCSK9 and bound SIRT1 iso-1 plasma from a healthy subject showing SIRT1 iso-1 is bound to PCSK9 in the plasma, (b) Representative western blot of acetyl-lysine and loading control, PCSK9 showing rSIRTI deacetylating rPCSK9 when incubated with its co-factor NAD+. (c) Schematic diagram of PCSK9 functional domains and an overview of PCSK9 acetylation. (d) Representative western blot of LDLR, SIRT1 , PCSK9 and β-actin expression in Huh7 cells expressing WT, 3KR, 3KQ mutants for 24h (n=3). (e) Representative western blot of SIRT1 , PCSK9, ApoB and ApoA1 on lipid subfractions (VLDL, LDL and HDL) from normolipidemic healthy subject. One representative fraction was taken for western blotting from FPLC of lipid sub-fractions from plasma (n=1 ). (f) Representative western blot of LDLR, SIRT1 , PCSK9 and β-actin expression in liver lysates isolated from wild-type and LDLR knockout mice treated with vehicle and rSIRTI for one hour (n=3). (g) Representative western blot of LDLR, SIRT1 , PCSK9 and β-actin expression in Huh7 cells overexpressing EGF-AB domain and EGF-AB(H306Y) mutant domain of LDLR and treated with rSIRTI , showing an increase in rSIRTI uptake upon EGF-AB or EGF-AB (H306Y) mutant domain expression (n=3). Veh, vehicle; rSIRTI , recombinant SIRT1 iso-1 ; 3KR, deacetyl mimetic PCSK9 with mutations at K243R/K421 R/K506R; 3KQ, acetyl mimetic PCSK9 with mutations at K243Q/K421 Q/K506Q; EGF-AB, epidermal growth factor-AB; AU, arbitrary units.
Fig. 3 shows that recombinant SIRT1 treatment protects against atherosclerosis and reduces plasma LDL-cholesterol in ApoE-/- mice. Ten week-old ApoE-/- mice were fed a high cholesterol diet (1.25% w/w) and treated with rSIRTI (n=6) or with PBS (n=6) for 10 weeks. Representative pictures (left) and quantifications (adjacent bar graph) of thoracoabdominal aortae en face (a) and of aortic root cross sections (b, c) stained with ORO or immunohistochemically for macrophages (CD68); (d) Graph showing cholesterol distribution in the different lipoprotein sub-fractions separated by gel filtration chromatography; (e) Bar graph of plasma total cholesterol and LDL-cholesterol concentrations, (f) Bar graph of ELISA of plasma PCSK9 and acetylated PCSK9 (g) Western blot of LDLR, SIRT1 , PCSK9 and β- actin in liver lysates (h) Bar graph of the relative gene expression of SIRT1 , LDLR and PCSK9. Scale bars in photomicrographs: 1 mm (a) and 500 mm (b, c). Veh, vehicle; rSIRTI , recombinant SIRT1 iso-1 ; AU, arbitrary units; ORO, oil-red O; LDL, low-density lipoprotein.
Fig. 4 shows a correlation of plasma SIRT1 and human pathophysiology, (a) Bar graph of an ELISA of plasma SIRT1 levels in healthy subjects of different age groups. Each bar represents 10 years beginning with 21 years of age with an n=20 per decade, (b) Representative correlation graph of SIRT1 and LDL-C levels in plasma of healthy subjects.
Fig. 5 shows that SIRT1 is secreted in plasma and modulates gene expression, (a) Relative gene expression of genes involved in glucose and lipid metabolism after treatment with rSIRTI for 8h in the presence of serum (24h) (n=8 per group). Gene expression is normalized to Rps29. (b, c) Representative bar graph of secreted from (b) GFP-tagged SIRT1 iso-1 or (c) GFP-tagged SIRT1 iso-2 secreted overexpressing Huh7 hepatoma cells compared to empty vector (E.V.) controls treated with the indicated classical and non- classical secretory pathway inhibitors (n=8 per group), (d) Western blot of SIRT1 expression in plasma samples of normolipidemic healthy subjects (n=3). Veh, vehicle; SIRT1 iso-1 , SIRT1 isoform 1 ; SIRT1 iso-2, SIRT1 isoform 2; rPCSK9, recombinant PCSK9; E.V., empty vector; AU, arbitrary units; scr, scramble.
Fig. 6 shows that SIRT1 modulates LDLR expression and PCSK9 activity, (a) Western blot of LDLR, SIRT1 , PCSK9 and β-actin expression in Huh7 cells incubated with SIRT1 at the indicated time-points (b) Relative mRNA expression of LDLR and PCSK9 24h after addition of rSIRTI (c) Representative dose-response of the binding activity of acetylated and deacetylated PCSK9 and LDLR EGF-A. (d, e) Representative western blot of LDLR, SIRT1 , PCSK9 and β-actin expression in Huh7 cells in the (d) absence of serum and (e) presence of serum, overexpressing SIRT1 iso-1 or empty vector control and treated exogenously with active rPCSK9 (5ng/ml) for 1 h showing a decrease in LDLR expression in empty vector control cells and a lack thereof in SIRT1 overexpressing cells, (f) Representative western blot of LDLR, SIRT1 , PCSK9 and β-actin expression in cell lysates of Huh7 cells treated with scramble siRNA as control and LDLR siRNA and incubated with vehicle or rSIRTI for one hour. Veh, vehicle; rSIRTI , recombinant SIRT1 iso-1 ; rPCSK9, recombinant PCSK9; E.V., empty vector; AU, arbitrary units; scr, scramble.
Fig. 7 shows identification and mapping of PCSK9 (SEQ ID NO 033) acetylation and deacetylation by SIRT1 . Recombinant PCSK9 was incubated with recombinant SIRT1 with and without NAD+. The protein from the Colloidal Coomassie stained band was digested with trypsin and the resulting peptides were analyzed by LC-MS/MS and MALDI MS/MS. MS/MS data were analyzed using the sequences of PCSK9 with the Mascot algorithm, allowing the detection of acetylated lysine residues, (a) MS/MS spectrum of all peptides recovered in the LC-MS/MS experiment for PCSK9, highlighted in yellow and lysine modification highlighted by green (b) Mass spectra obtained from the recombinant protein lysate digested with trypsin and subjected to LC-MS/MS analysis (c) MS/MS spectrum of all peptides recovered in the LC-MS/MS experiment for PCSK9 after performing PCSK9 deacetylation assay, highlighted in yellow and lysine modification highlighted by green Acetylation was detected lysine residue 243, 273, 421 and 506 in the fragmentation spectrums of peptides from trypsin- digested PCSK9. In the presence of SIRT1 and co-factor NAD+ acetylation of only lysine
residue 273 was detected, but not of lysine residues 243, 421 and 506 in the fragmentation spectrums of peptides from trypsin-digested PCSK9.
Fig. 8 shows that Recombinant SIRT1 treatment reduces macrophage accumulation reduces and plasma VLDL-cholesterol in ApoE-/- mice. Ten week-old ApoE-/- mice were fed a high cholesterol diet (1.25% w/w) and treated with rSIRTI (n=6) or with PBS (n=6) for 10 weeks. Representative pictures immunohistochemically stained with H&E. Marcrophages on liver (a) and kidney (b) indicated with black arrow; (c, d) Bar graph of plasma VLDL cholesterol and HDL-cholesterol concentrations. (Scale bars in photomicrographs: 1 mm (a) and 500 mm (b, c). Veh, vehicle; rSIRTI , recombinant SIRT1 iso-1 ; AU, arbitrary units; VLDL, very low- density lipoprotein; HDL, high-density lipoprotein.
Fig. 9 shows the effect of recombinant SIRT1 treatment in ApoE-/- mice. Ten week-old ApoE-/- mice were fed a high cholesterol diet (1 .25% w/w) and treated with rSIRTI (n=6) or with PBS (n=6) for 10 weeks, (a) Graph body weight course measured every week for the duration of the treatment, (b) average daily food intake (d) and weight of epididymal white adipose tissue (epiWAT) (b, c). Veh, vehicle; rSIRTI , recombinant SIRT1 iso-1 ; epiWAT, epididymal white adipose tissue.
Fig. 10 shows the effect of recombinant SIRT1 treatment on glucose metabolism in ApoE-/- mice. Ten week-old ApoE-/- mice were fed a high cholesterol diet (1.25% w/w) and treated with rSIRTI (n=6) or with PBS (n=6) for 10 weeks, (a) Bar graph of non-fasting glucose and fasting glucose measured at the end of 12 -weeks of treatment. Mice were fasted for 6h before measurement (c, d) Representative pictures (left) and quantifications (adjacent bar graph) of pancreatic islets with (c) insulin as a surrogate maker for β-cells and with (d) glucagon as a surrogate maker for a-cells. Positive area normalised to total area of islets. Scale bars in photomicrographs: 1 mm (a) and 500 mm (b, c). Veh, vehicle; rSIRTI , recombinant SIRT1 iso-1 ; AU, arbitrary units.
Fig. 1 1 shows the correlation of plasma SIRT1 and and PCSK9 in healthy subjects (a) Representative correlation log graph of SIRT1 and PCSK9 levels in plasma of healthy subjects of different age groups (n= 80, r= -0.4419, p < 0.001 ). Data presented as log graph, (b) Representative correlation graph of SIRT1 and PCSK9 levels in plasma of patients with myocardial infarction (n= 184, r= -0.1534, p = 0.0439). Data presented as log graph.
Fig. 12 shows that rSIRTI treatment improves insulin sensitivity in wild-type mice on high-fat diet. Insulin tolerance test was performed in fasted wild-type mice were fed high-fat diet for 4 months and treated with vehicle or recombinant SIRT1 (rSIRTI ) for one month. Mice were injected i.p. with insulin in fasted mice. Blood glucose levels were determined at the indicated time points. Values are expressed as means ± s.e.m. (n=10 per group), *p<0.05.
Fig. 13 shows that rSIRTI treatment improves glucose sensitivity in wild-type mice on high- fat diet. Glucose tolerance test was performed in fasted wild-type mice were fed high-fat diet for 4 months and treated with vehicle or recombinant SIRT1 (rSIRTI ) for one month. Mice were injected i.p. with glucose in fasted mice. Blood glucose levels were determined at the indicated time points. Values are expressed as means ± s.e.m. (n=10 per group), *p<0.05.
Fig. 14 shows that rSIRTI treatment reduces body-weight in wild-type mice on high-fat diet. Wild-type mice were fed high-fat diet for 4 months and treated with vehicle or recombinant SIRT1 (rSIRTI ) for one month. Body-weight were measured on a weekly basis for the duration of the treatment. Values are expressed as means ± s.e.m. (n=10 per group), *p<0.05.
Fig. 15 shows that rSIRTI treatment increases liver weight in ApoE"'" mice. Ten week-old ApoE"'" mice were fed a high cholesterol diet (1.25% w/w) and treated with rSIRTI (n=7) or with PBS (n=7) for 10 weeks. Bar graph representing the epididymal white adipose tissue (epiWAT) and liver weight of ApoE"'" mice treated with rSIRTI compared to vehicle (PBS) controls. Values +/- SEM.
Fig. 16 shows that rSIRTI treatment tends to reduce steatohepatitis in ApoE"'" mice. Ten week-old ApoE"'" mice were fed a high cholesterol diet (1 .25% w/w) and treated with rSIRTI (n=7) or with PBD (n=7) for 10 weeks. Representative pictures (left) and quantifications (adjacent bar graph) of liver cross sections staining with Oil-red O dye.
Fig. 17 shows that SIRT1 circulates in the plasma. Representative western blot of SIRT1 immunoprecipitated from plasma of healthy subjects.
Fig. 18 shows the time-course of SIRT1 treatment (2 pg) in Huh7 cells. Representative western blot of SIRT1 , LDLR, PCSK9 and β-actin of Huh7 cells treated with recombinant SIRT1 .
Fig. 19 shows that rSIRTI treatment reduces plasma lipids in ApoE"'" mice. Ten week-old ApoE"'" mice were fed a high cholesterol diet (1.25% w/w) and treated with rSIRTI (n=6) or with PBS (n=6) for 10 weeks. Bar graphs of plasma total-cholesterol, LDL-cholesterol, HDL- cholesterol and VLDL-cholesterol. Values +/- SD.
Fig. 20 shows that rSIRTI treatment does not alter plasma PCSK9 levels of ApoE"'" mice. Ten week-old ApoE"'" mice were fed a high cholesterol diet (1.25% w/w) and treated with rSIRTI (n=7) or with PBS (n=7) for 10 weeks. Bar graphs of ELISA of plasma PCSK9 protein.
Examples
Sirtuin 1 (SIRT1 ) is a member of the Sirtuin family of NAD+- dependent deacetylase enzymes and an important regulator of nutrient metabolism and aging. Recently, several studies have attempted to detect SIRT1 in the plasma of different patient cohorts. Thus the inventors decided to investigate whether SIRT1 is secreted by hepatocytes and if so, to which of the two isoforms of SIRT1 this phenomenon be attributed to. To this end, the inventors performed isoform-specific overexpression of GFP-tagged SIRT1 in Huh7 cells and analysed the secretion of SIRT1 using a GFP ELISA (Fig. 1 a). Surprisingly, both isoforms were secreted, however, to varying degrees. Indeed, SIRT1 isoform 1 (iso-1 ) was secreted to a substantially higher degree suggesting that iso-1 is the dominant secreted isoform. Since SIRT1 is involved in different metabolic pathways, the inventors determined, if isoform- specific SIRT1 differentially regulates intracellular metabolic pathways. The inventors found five genes to be differentially regulated by the two isoforms. A significantly higher expression of SLC2A4 and FASN was observed in SIRT1 iso-2 overexpressing Huh7 cells compared to empty vector and SIRT1 iso-1 (Fig. 1 b). Correspondingly, SIRT1 iso-1 decreased the expression of HK2 and PCK1 . To distinguish between genes modulated by intracellular and extracellular SIRT1 iso-1 , and extracellular activity of SI RT1 iso-1 alone, the inventors added recombinant SIRT1 iso-1 (rSIRTI ) to Huh7 cells (Fig. 1 c). Addition of rSIRTI in the absence of serum significantly reduced HMGCR expression suggesting a regulatory role in lipid metabolism. In the presence of serum, rSIRTI increased FABP4 suggesting that secreted SIRT1 alone has a different but significant in metabolic homeostasis compared to intracellular SIRT1 . Intracellular SIRT1 is induced upon nutrient deprivation, while not being transcriptionally increased. Consequently, the inventors investigated the metabolic conditions under which SIRT1 isoforms may be secreted by overexpressing GFP-tagged SIRT1 isoform secretion under varying amounts of serum (Fig. 1 d). Surprisingly, reducing serum levels decreased the secretion of both isoforms in a concentration-dependent manner. The inventors used different inhibitors of the classical and non-classical secretory pathway to assess the mechanism of secretion. They found no change in SIRT1 secretion upon addition of classical or non-classical inhibitors Fig. 5b and c. To confirm that SIRT1 is indeed secreted in humans, the inventors performed a western blot analysis of SIRT1 in plasma samples from normolipidemic healthy subjects. A 1 10k Da band specific to SIRT1 iso-1 was detected western blotting of plasma samples (Fig. 5d). Taken together, these results suggest a complex, but important role of isoform-specific SIRT1 secretion in the homeostatic regulation of lipid metabolism in the liver.
Previously the inventors have shown that pharmacological activation of SIRT1 causes a decrease in LDL-C. Thus, they assessed the involvement of secreted SIRT1 iso-1 in modulating hepatic LDLR expression. rSIRTI increased LDLR protein expression in a time-
dependent manner with a maximal effect at 8 h after addition (Fig. 6a). At this point it could not be excluded that the gradual rise in LDLR expression is due to the fact that rSIRTI has used NAD+ produced by the cell (as NAD+ was not added externally). The inventors further observed a time-dependent manner increase in SIRT1 expression, suggesting an uptake of SIRT1 . LDLR expression is regulated post-transcriptionally by plasma PCSK9. rSIRTI did not affect gene expression of LDLR or PCSK9 at 8 h compared to vehicle (Fig. 6b), thus prompting us to examine if SIRT1 iso-1 directly binds to PCSK9 while in circulation. Pulldown experiments from normolipidemic human plasma revealed that SIRT1 was indeed bound to PCSK9, thus implying that SIRT1 may directly deacetylate PCSK9 in plasma (Fig. 2a). To confirm if SIRT1 iso-1 directly deacetylates PCSK9, recombinant PCSK9 (rPCSK9) and rSIRTI were incubated with or without co-factor NAD+ in vitro and analysed by Western blot analysis. In samples incubated with NAD+, levels of acetyl-lysine were reduced compared to those without NAD+ (Fig. 2b), further confirming that PCSK9 is a substrate of SIRT1 .
The number and location of PCSK9 acetylation sites are yet unknown. Mass spectrometry of PCSK9 immunoprecipitated from Huh7 cells, revealed a total of 4 unique acetylation sites, 3 in the catalytic domain and one in the C-terminal domain - Lys243, Lys273, Lys421 and Lys506, respectively (Fig. 2c, Fig. 7a and b). Incubation of PCSK9 with rSIRTI and NAD+ caused deacetylation at 3 of 4 acetylation sites (Fig. 7c). SIRT1 iso-1 did not deacetylate Lys273. To evaluate the functional importance of acetylation at Lys243, Lys421 and Lys506 on PCSK9, the inventors generated triple mutants, mutating lysine groups to glutamine to mimic acetylation (K243Q/K421 Q/K506Q) (PCSK9-3KQ) or mutating lysine groups to arginine to mimic deacetylation (K243R/K421 R/K506R) (PCSK9-3KR). Overexpression of acetylation mimetic, PCSK9-3KQ, mimicked the effect of wild-type PCSK9 in reducing LDLR expression in Huh7 cells, while deacetylation mimetic PCSK9-3KR failed to reduce LDLR expression (Fig. 2d), suggesting a decrease in activity. To further extend their findings in an in vitro model, the inventors assessed if the acetylation status of PCSK9 affects binding of PCSK9 to the LDL-R epidermal growth factor (EGF) A-domain using a PCSK9-LDLR binding assay. Human rPCSK9 incubated with rSIRTI and NAD+ (Fig. 6b) showed a reduced binding of PCSK9 to LDLR EGF(A)-domain in the deacetylated form compared to the acetylated form of PCSK9, in a concentration-dependent manner, suggesting that deacetylated PCSK9 has reduced activity. Subsequently, the inventors explored the effects of addition of PCSK9 to SIRT1 iso-1 overexpressing cells. They transfected SIRT1 iso-1 or empty vector in Huh7 cells and incubated them with rPCSK9 for an hour in the absence or presence of serum (Fig. 6c, d). In the absence of serum, rPCSK9 reduced LDLR expression in control cells, but failed to reduce LDLR expression in SIRT1 overexpressing cells. In the presence of serum rPCSK9 failed to reduce LDLR expression in
empty vector controls and SIRT1 overexpressing cells (Fig. 6d). However, the internalisation of rPCSK9 increased upon SIRT1 overexpression, suggesting that deacetylation of PCSK9 affects only the targeting of LDLR, but not PCSK9 internalisation. Although the inventors provide evidence that circulating SIRT1 interacts with circulating PCSK9 in plasma, it remains unclear if SIRT1 is associated with any larger lipoprotein complexes in plasma, similar to circulating PCSK9. To address this, SIRT1 was binding was assessed in lipoproteins isolated from human plasma from normolipidemic healthy subjects by FPLC. The inventors found SIRT1 to be present in the HDL sub-fractions indicating that SIRT1 is bound to HDL particles while in circulation (Fig. 2e). Similar to other studies, they found PCSK9 to be dominantly bound to HDL in human plasma. This fraction contained the majority of apoA1 and undetectable levels of ApoB. Thus implying that PCSK9 and SIRT1 may interact via HDL.
Earlier findings showed that SIRT1 also increased in an LDL-R-dependent manner, prompting the inventors to address if SIRT1 binds LDLR similar to PCSK9. To confirm if rSIRTI is indeed internalised via the LDLR, they incubated Huh7 cells with rSIRTI upon LDLR knockdown (Fig. 6f). rSIRTI did not increase upon LDLR knockdown compared scramble control. Accordingly, in vivo, rSIRTI was not internalised in LDLR KO mice compared to wild-type mice, which showed a clear increase in SIRT1 iso-1 expression upon addition of rSIRTI (Fig. 6f). PCSK9 binds to the EGF-AB domain of LDLR. Since PCSK9 is a substrate of SIRT1 and LDLR is the receptor of SIRT1 , it may be possible that SIRT1 binds to the same EGF-AB domain of LDLR, similar to PCSK9. To test this hypothesis in vitro, the inventors overexpressed EGF-like repeats A and B (EGF-AB) and an EGF-AB domain mutated at His-306 (EGF-AB (H306Y)). His-306 of the EGF-AB is replaced by tyrosine in the FH-associated H306Y LDLR allele and exhibits 2.5-fold increased binding affinity to PCSK9. Huh7 cells transfected with empty vector, EGF-AB domain or EGF-AB (H306Y) were incubated 24h post-transfection with 5 pg rSIRTI for 1 h. rSIRTI internalisation was higher upon EGF-AB overexpression compared to empty vector. Overexpression of EGF-AB (H306Y) was even higher, suggesting that PCSK9 and SIRT1 iso-1 bind to the same EGF- AB domain on LDLR and compete with each other for internalisation These novel findings indicate that SIRT1 iso-1 is a modulator of PCSK9, is bound to HDL in plasma and that LDLR is a receptor of plasma SIRT1 iso-1 . Thus, circulating SIRT1 iso-1 is a novel regulator of hepatic cholesterol metabolism.
To ascertain a causal role of circulating SIRT1 iso-1 in regulating plasma LDL-C and in turn atherosclerosis, the inventors investigated the effects of alternate-day intraperitoneal injections of rSIRTI or vehicle in ApoE"'" mice, fed a high-cholesterol diet, over a period of 10 weeks. Compared to vehicle-treated ApoE"'" mice, en face preparations of the thoracic- abdominal aortas of rSIRTI-treated ApoE"'" mice showed a markedly reduced plaque area
(fig. 3a). Atherosclerotic plaque formation is driven by an accumulation of cholesterol-laden macrophages in the artery wall. Aortic root lesions of rSIRTI -treated ApoE"'" mice showed a significant reduction in plaque area along with macrophage accumulation (Fig. 3b and 3c) compared with vehicle-treated ApoE"'" mice. Of note, the inventors found an overall decrease in foam cell formation in the liver and kidneys of rSIRTI -treated mice compared to vehicle- treated control mice (Fig. 8a, b).
Since SIRT1 iso-1 modulates PCSK9, the inventors assessed plasma lipid sub-fractions between the two groups (Fig. 3d). In the rSIRTI -treated group they found a marked decrease in plasma total-cholesterol, LDL-C and VLDL-C (Fig. 3e, 3f, Fig. 8c), with no change in HDL- C (Fig. 8d) compared to vehicle-treated controls. The inventors found a significant increase in deacetylated PCSK9 in rSIRTI -treated mice with no difference in plasma PCSK9 levels between the two groups; suggesting that SIRT1 iso-1 does not affect PCSK9 expression, but its activity (Fig. 3g and 3h). Accordingly, they observed a significant increase in hepatic LDLR and SIRT1 protein expression, with no change in hepatic PCSK9 protein expression (Fig. 3i). Quantitative PCR analysis, showed no change in LDLR and PCSK9 gene expression but reduced SIRT1 gene expression, implying a negative feedback of rSIRTI - treatment and rSIRTI uptake (Fig. 3j).
Deficiency for Sirtl in wild-type mice has previously been shown to reduce body weight while activation of Sirtl in ApoE"'" mice reduces body weight. Unlike the effect of SIRT1 activation in ApoE"'" mice, the inventors observed no difference in body weight, food intake or epididymal white adipose tissue weight between the two groups (Fig. 9a-c). Since SIRT1 modulates glucose metabolism and improves insulin sensitivity, the inventors measured plasma glucose levels which in the non-fasting state were significantly lower in rSIRTI - treated ApoE"'" mice (Fig. 10a), and a significant increase in plasma insulin levels (Fig. 10b). PCSK9 deficiency in pancreatic islets of mice caused a two-fold increase in LDLR protein expression in pancreatic islets, mainly beta cells. To assess if the improvement in insulin secretion by rSIRTI treatment is due to a greater number of β-cells, the inventors quantified β-cell and a-cell numbers using insulin as a surrogate marker for β-cells in islets and glucagon as a surrogate marker for a-cells in islets, respectively. They found a significant increase in the number of β-cells, with no difference in a-cells between the two groups (Fig. 10d, e). Thus, rSIRTI exhibits an increase in β-cell islet biogenesis. Consequently, the effects of rSIRTI on tissues other than liver such as the pancreatic β-cells may contribute to the reduced atherosclerosis in ApoE"'" mice. Thus, recombinant SIRT1 protects against atherosclerosis by decreasing plasma LDL-C and by improving glucose metabolism.
To explore a potential role of circulating SIRT1 in human physiology, the inventors measured plasma SIRT1 levels in healthy subjects of different age groups. Importantly, they
found an age-dependent decrease in circulating SIRT1 in healthy subjects (Fig. 4a) which inversely correlated to plasma LDL-C (Fig. 4b). Furthermore, SIRT1 significantly correlated with non-HDL cholesterol and ApoBI OO plasma levels (Table 1 ). Consequently, the inventors found a significant inverse correlation between plasma SIRT1 and PCSK9 levels (Fig. 1 1 a), implicating that circulating SIRT1 is an age-dependent marker of lipid homeostasis and that the age-dependent decrease in SIRT1 contributes to the age-dependent increase in plasma LDL-C and risk to cardiovascular disease. The inventors extended their findings further to dyslipidemia, i.e. in a cohort of non-statin treated obese, but non-diabetic individuals. They found a significant inverse correlation between circulating SIRT1 and plasma PCSK9 and LDL-C levels. Myocardial infarction is the final result of high LDL and in turn plaque formation. Of note, in an acute setting of infarction, plasma LDL-C decline rapidly within 24h post-MI. Recently high levels of PCSK9 have been associated with an increase in inflammation in the acute phase of myocardial infarction in a large Swiss multicentre cohort study.
In summary, the inventors here provide the first evidence that SIRT1 is secreted and that circulating SIRT1 regulates plasma PCSK9 activity by deacetylating specific acetylation sites on the protein and in turn reduces LDL-C and atherosclerotic plaque size and composition. Furthermore, the inventors show that SIRT1 is bound to HDL in circulation, binds to hepatic LDLRs and thereby cleared from circulation. Finally, recombinant SIRT1 provides a novel cardiovascular treatment modality to reduce plaque size and composition and to improve glucose homeostasis.
Table 1. Correlation between plasma SIRT1 and selected metabolic parameters in healthy subjects of different age groups.
Patients n=80 r p
Age, years -0.3918 0.0214*
Total cholesterol, mmol/l -0.3171 <o.ooor
HDL cholesterol, mmol/l -0.04653 0.6839
non-HDL cholesterol, mmol/l -0.2902 0.0095*
Triglycerides, mmol/l -0.2494 0.0266*
Lipoprotein A 0.08749 0.4433
Mice
Male ApoE"'" mice on a pure C57BL/6J background, genotyped as per standardized protocol, were housed with a 12 h light-dark cycle and fed a high-cholesterol diet containing 1.25% cholesterol (D 12108; Research Diets) with alternate day injections of PBS (vehicle) and recombinant SIRT1 (10 pg per injection). After this treatment period, mice were sacrificed and tissues were harvested. All experiments and animal care procedures were approved by the local veterinary authorities and carried in accordance with our institutional guidelines.
Cell Culture
Huh7, human hepatoma cells were cultured in DMEM (Sigma) supplemented with Glutamax. Where indicated cells were treatment with PBS (vehicle) or recombinant SIRT1 (500 ng) for a period of 6 hours or 1 hour. Where indicated cells were treated with cycloheximide, Brefeldin A or Methylamine for a period of 1 hour. LDH assay for toxicity was performed using manufacturers protocol. Recombinant active PCSK9 (Cusabio, Abeam) for cell culture were analysed by mass spectrometry and used for uptake analysis at indicated concentrations.
Cholesterol profile in the different lipoprotein fractions
Total cholesterol plasma concentration is measured using a colorimetric method (BioMerieux, France). Plasma sample is loaded on a gel chromatography column, which separates lipoproteins according to their size. The chromatograph effluent is immediately and continuously mixed with cholesterol assay reactant, and incubated at the appropriate conditions of temperature and duration to allow the enzymatic reaction to occur. The OD measured at the appropriate wavelength is automatically converted in a graphic signal representing the lipid distribution profile. The area under each peak is proportional to the lipid concentration in the respective lipoprotein fraction.
LDLR and PCSK9 binding assay
PCSK9-LDLR interaction was assessed using a CircuLex PCSK9-LDLR in vitro binding assay kit (MBL International, Woburn, MA, USA) with minor modifications. Recombinant His- tagged PCSK9 (1 pg/mL) or recombinant PCSK9 at a concentration of 100 pg/mL were subject to a deacetylation assay for 1 h at room temperature with gentle shaking. The mixtures were added to an ELISA plate that was coated with EGF-AB peptide of the LDLR. Subsequent procedures were performed according to the manufacturer's instructions. Where required the detection antibody was replaced with his-tag antibody and anti-PCSK9 antibody.
Western blotting
Total protein from tissues and cells was prepared as described and analysed using standard protocols.10 Protein samples were immunostained using specific antibodies targeted to SIRT1 (Cell signalling), LDL-R (Novus Biologicals), PCSK9 (Abeam) or β-actin, and detected by horseradish peroxidase chemiluminescence.
Real-time quantitative PCR
Total RNA was extracted using Trizol reagent (Sigma-Aldrich) according to the manufacturer's protocol. RNA was reverse transcribed with random hexamers and the cDNA obtained was quantified by SYBR-green qPCR using gene-specific primers. Results were normalized to Rps29.
Plasmids and vectors
SIRT1 isoform 1 (EX-U1443-M29, Genecopoeia), SIRT1 isoform 2 (EX-Z6705-M29, Genecopoeia) and wild-type PCSK9 (RC220000, Origene) were transfected using Lipofectamine 3000 following manufacturers protocol. Cells were harvested 24h post- transfection.
Deacetylation assay
Recombinant PCSK9 was incubated with recombinant SIRT1 in a buffer (250mM Tris-HCI, 20mM MgCI2, 250mM NaCI) in the presence and absence of 5mM NAD+ for one hour at 37°C. The reaction was stopped using laemmeli buffer and subject to western blotting.
LDL-cholesterol uptake and flow cytometry
Huh7 cells were cultured in a six-well plate and incubated with recombinant SIRT1 at 500ng concentration for 1 h. After treatment, cells were washed twice with phosphate-buffered saline and then incubated with 5 pg/ml of Bodipy-labelled human LDL (L-3483, Invitrogen) in serum-free medium for 1 h at 37 °C. After washing twice with phosphate-buffered saline, uptake of Bodipy-LDL-C was determined by flow cytometry.
Immunoprecipitation
PCSK9 antibody (RnD Systems) was covalently linked to beaded agarose resin via primary amines using the Aminolink Plus immobilization kit (Pierce, catalog number 44894). Serum samples (200 μΙ) were then diluted 5-fold in immunoprecipitation buffer [50 mM HEPES (pH 7.40), 150 mM NaCI, 1 % Triton X-100, 5 mM EDTA, and 5 mM EGTA] for a total of 1 ml volume and incubated overnight with 20 pg of PCSK9 antibody linked to 40 μΙ packed beads. The beads were washed five times with PBS + 0.1 % NP-40 used for Western blotting analysis.
ELISA
Mice were fasted over night before blood was drawn prior to harvesting. For plasma analyses EDTA plasma was separated from corpuscular elements by centrifugation at 4 °C immediately after blood was drawn and stored at - 80 °C until analysis. TNFa, IL-6, and MCP-1 levels in plasma samples were measured (Millipore). ELISA for mouse PCSK9 in the plasma was performed using Mouse ELISA kit (RnD Systems). ELISA for GFP (Abeam), human SIRT1 (Cusabio) and PCSK9 (RnD Systems) was performed using manufacturers protocol with appropriate dilutions (no dilution for cell culture supernantant, 1 :2 for SIRT1 and 1 :20 for PCSK9). All ELISA analysis was performed in samples without freeze-thaw.
Immunohistochemistry
Serial cryosections from the aortic sinus and liver were obtained from frozen tissues fixed in OCT, while pancreas and muscle were fixed in 4% formaldehyde. Cryosections were fixed in acetone and all sections were blocked in 2% normal mouse serum. They were stained with oil-red O (ORO), anti-CD68 (Abeam), VCAM-1 (Abeam) antibodies for the aortae, ORO, anti- LDLR for liver and haematoxycilin for skeletal muscle. Thoraco-abdominal aortae were fixed with 4% paraformaldehyde and plaques were stained with ORO for en face analysis of atherosclerotic lesion area and analysed as described.
Mass spectrometry
Excised gel bands were cut into approximately 1 mm3 pieces. Gel pieces were washed twice with 100 mM ammonium bicarbonate/50% acetonitrile for 15 min at 50°C and dehydrated with acetonitrile for 10 min. All supernatants were discarded. Rehydration of the gel pieces was with 10 mM Tris-HCI, 2 mM CaCI2, pH 8.2 containing 5 ng/μΙ proteomics-grade recombinant trypsin (Roche, Diagnostics, Mannheim, Germany) at 4°C. Microwave assisted digestion (Model Discover, CEM, Matthews, NC) was performed for 30 min at 5 W and 60 C. The supernatant was extracted and gel pieces were washed with 150 ul 0.1 % trifluoroacetic acid/50% acetonitrile. All supernatants were combined and evaporated to dryness in a SpeedVac concentrator. Digested samples were re-solubilized in 20 uL of 0.1 % formic acid and were analyzed by either MALDI time-of-flight tandem mass spectrometry MALDI-TOF- TOF) or liquid chromatography electrospray tandem mass spectrometry (LC/MS/MS For Maldi-TOF-TOF 1 ul of the sample was mixed with 1 ul of matrix solution (0.7 mg/ml a-cyano- 4-hydroxycinnamic acid in 0.1 % trifluoroacetic acid/85% acetonitrile, 1 mM NH4H2PO4) and spotted on the target, desalted and concentrated by washing the spot with 0.1 % trifluoroacetic acid. Maldi spectra were acquired on an UltrafleXtreme (Bruker, Bremen, Germany). LC/MS/MS analyses were run on a nanoAcquity UPLC (Waters Inc.) connected to a Q Exactive mass spectrometer (Thermo Scientific) equipped with a Digital PicoView source
(New Objective). An aliquot of 2 [L was injected. Peptides were trapped on a Symmetry C18
trap column (5 pm, 180 pm x 20 mm, Waters Inc.) and separated on a BEH300 C18 column (1 .7 pm, 75 pm x 150 m, Waters Inc.) at a flow rate of 250 nl/min using a gradient from 1 % solvent B (0.1 % formic acid in acetonitrile, Romil)/99% solvent A (0.1 % formic acid in water, Romil) to 40% solvent B/60% solvent A within 90 min. Full scan MS spectra were acquired in positive profile mode from 350 - 1500 m/z with an automatic gain control target of 3e6, an Orbitrap resolution of 70Ό00 (at 200 m/z), and a maximum injection time of 100 ms. The 12 most intense multiply charged (z≥ +2) precursor ions from each full scan were selected for higher-energy collisional dissociation fragmentation with a normalized collision energy of 25 (arbitrary unit). Generated fragment ions were scanned with an Orbitrap resolution of 35Ό00 (at 200 m/z) an automatic gain control value of 1 e5 and a maximum injection time of 120 ms. The isolation window for precursor ions was set to 2.0 m/z and the underfill ratio was at 3.5% (refereeing to an intensity threshold of 2.9e4). Each fragmented precursor ion was set onto the dynamic exclusion list for 40 s.
Peptides were identified by aligning the Maldi-TOF-TOF data with the known sequence of PCSK9 using the BioTools program (Bruker, Bremen, Germany) or by searching the SwissProt database (version 2015_1 1 , 549832 entries) using the Mascot search engine (Matrix Science, version 2.4.1 ). Mascot was set up to search the SwissProt database assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.030 Da and a parent ion tolerance of 10.0 PPM. Oxidation of methionine and acetylation of lysine was specified in Mascot as a variable modification. Scaffold (version Scaffold_4.4.8, Proteome Software Inc.) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they achieved an FDR less than 0.1 % by the Scaffold Local FDR algorithm. Protein identifications were accepted if they achieved an FDR less than 1.0% and contained at least 2 identified peptides.
Healthy subjects
Plasma from eighty subjects were used in the study. None of the subjects took prescription medicines. Blood samples were obtained from Biutspende Zurich. Twenty subjects were considered between the age groups - 21 to 30 years, 31 to 40 years, 41-50 years and 51-60 years. Lipid profile measurements were performed at University Hospital Zurich.
Statistics
All data are presented as the mean ± SEM and were analyzed by unpaired two-tailed Student's t test or a non-parametric test or one-way ANOVA with Tukey comparison test or multiple comparison t-test where appropriate. At least three independent experiments in triplicates were performed. *P < 0.05 and **P < 0.01 were considered significant. Analyses were done using Graphpad Prism version 6.0d 2010.
Sequences
SEQ ID NO 01 Sirtl isoform 1
MADEAALALQPGGSPSAAGADREAASSPAGEPLRKRPRRDGPGLERSPGEPGGAAPEREVPA AARGCPGAAAAALWREAEAEAAAAGGEQEAQATAAAGEGDNGPGLQGPSREPPLADNLYDED DDDEGEEEEEAAAAAIGYRDNLLFGDEI ITNGFHSCESDEEDRASHASSSDW PRPRIGPYT FVQQHLMIGTDPR ILKDLLPE I PPPELDDMTLWQIVINILSEPPKRKKRKDIN IEDAVK LLQECKKI IVLTGAGVSVSCGI PDFRSRDGIYARLAVDFPDLPDPQAMFDIEYFRKDPRPFF KFAKEIYPGQFQPSLCHKFIALSDKEGKLLRNYTQ IDTLEQVAGIQRI IQCHGSFA ASCL ICKYKVDCEAVRGDIFNQVVPRCPRCPADEPLAIMKPEIVFFGENLPEQFHRAMKYDKDEVD LLIVIGSSLKVRPVALI PSS I PHEVPQILINREPLPHLHFDVELLGDCDVI INELCHRLGGE YAKLCCNPVKLSEI EKPPRTQKELAYLSELPPTPLHVSEDSSSPERTSPPDSSVIVTLLDQ AAKSNDDLDVSESKGCMEEKPQEVQ SRNVES IAEQMENPDLKNVGSS GEKNER SVAGTV RKCWPNRVAKEQI SRRLDGNQYLFLPPNRYIFHGAEVYSDSEDDVLSSSSCGSNSDSGTCQS PSLEEPMEDESEIEEFYNGLEDEPDVPERAGGAGFG DGDDQEAINEAI SVKQEV DMNYPS NKS
SEQ ID NO 02 Sirtl isoform 2
MADEAALALQPGGSPSAAGADREAASSPAGEPLRKRPRRDGPGLERSPGEPGGAAPEREVPA AARGCPGAAAAALWREAEAEAAAAGGEQEAQATAAAGEGDNGPGLQGPSREPPLADNLYDED DDDEGEEEEEAAAAAIGYRDNLLFGDEI I NGFHSCESDEEDRASHASSSDWTPRPRIGPYT FVQQHLMIGTDPRTILKDLLPETI PPPELDDMTLWQIVINILSEPPKRKKRKDINTIEDAVK LLQECKKI IVLTGAGVSVSCGI PDFRSRDGIYARLAVDFPDLPDPQAMFDIEYFRKDPRPFF KFAKEIYPGQFQPSLCHKFIALSDKEGKLLRNYTQNIDTLEQVAGIQRI IQCHGSFATASCL ICKYKVDCEAVRGDIFNQVVPRCPRCPADEPLAIMKPEIVFFGENLPEQFHRAMKYDKDEVD LLIVIGSSLKVRPVALI PSNQYLFLPPNRYIFHGAEVYSDSEDDVLSSSSCGSNSDSGTCQS PSLEEPMEDESEIEEFYNGLEDEPDVPERAGGAGFGTDGDDQEAINEAI SVKQEVTDMNYPS NKS
SEQ ID NO 003 to SEQ ID NO 032 are listed in Table 2.
SEQ ID NO 33 human PCSK9
MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTA TFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLV KMSGDLLELALKLPHVDYIEEDSSVFAQS I PWNLERITPPRYRADEYQPPDGGSLVEVYLLD TSIQSDHREIEGRVMVTDFENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMR SLRVLNCQGKGTVSGTLIGLEFIRKSQLVQPVGPLVVLLPLAGGYSRVLNAACQRLARAGVV
LVTAAGNFRDDACLYSPASAPEVITVGATNAQDQPVTLGTLGTNFGRCVDLFAPGEDIIGAS SDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQR VLTPNLVAALPPSTHGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRR GERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHV LTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREAS IHASCCHAPGLECKVKEHGI PAPQE QVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSR HLAQASQELQ
Claims
1. An isolated polypeptide comprising an amino acid sequence characterized by at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, 96%, >97%, >98%, >99%, >99.5%, >99.9% sequence identity to SEQ ID NO 01 or SEQ ID NO 02, said isolated polypeptide having an NAD+-dependent deacetylase function identical to SIRT1 isoform 1 or isoform 2, for use in a method of treatment or prevention of a condition selected from the group comprising dyslipidemia, hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, diabetes, metabolic syndrome and obesity-related conditions.
2. An isolated polypeptide for use in a condition according to claim 1 , wherein said isolated polypeptide is recombinant SIRT1 isoform 1 or a polypeptide sequence having a sequence identity of at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, 96%, >97%, >98%, >99%, >99.5%, >99.9% sequence identity to SEQ ID NO 01 , said polypeptide sequence having at least 85% of the in-vitro an NAD+-dependent PCSK9 deacetylase activity of SIRT1 isoform 1 .
3. A dosage form comprising the isolated polypeptide according to claim 1 or 2 for use in therapy or prevention of a condition selected from the group comprising dyslipidemia, hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, diabetes, metabolic syndrome and obesity-related conditions.
4. The dosage form according to claim 3, wherein the amount of said isolated polypeptide is 1 pg to 5 mg per dose, particularly 10 pg to 5 mg, more particularly 100 pg to 3.5 mg, even more particularly 500 pg to 3.5 mg per dose.
5. The dosage form according to any one of claims 3 to 4, formulated for parenteral, peroral, transdermal or transmucosal administration.
6. A combination medicament comprising
a. the isolated polypeptide according to claim 1 or 2 and
b. a SIRT1 activator or NAD+ supplement, in particular a compound selected from niacin (vitamin B3), nicotinamide mononucleotide (NMN), nicotinamide ribosome (NR) and resveratrol.
7. A method for treatment of a patient diagnosed with a pathological plasma lipid level said method comprising administering to said patient an effective amount of an isolated polypeptide as specified in claim 1 or 2.
8. The method of claim 7, wherein the method comprises adjusting said lipid level to a range not associated with disease.
9. The method of claim 7, wherein the method comprises improving HDL functionality.
10. The method of claim 7, wherein the method comprises improving glucose sensitivity and insulin sensitivity.
1 1 . The method of claim 7, wherein the method comprises reducing body weight in patients diagnosed with obesity.
12. A method of treatment of a condition selected from the group comprising dyslipidemia, hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, diabetes, metabolic syndrome and obesity-related conditions, said method comprising administration of an isolated polypeptide as specified in claim 1 or 2.
13. A method of determining the risk of a patient to develop coronary artery disease (CAD), said method comprising quantifying the level of SIRT1 expression, particularly quantifying the level of SIRT1 protein, in a sample obtained from said patient, and using the obtained information during assessment of the risk of said patient to develop CAD, wherein it is indicative of a high risk to develop CAD if said SIRT1 level is below a predetermined threshold.
14. A method of determining the risk of a patient to develop CAD, said method comprising quantifying the level of SIRT1 expression, particularly quantifying the level of SIRT1 protein, in a sample obtained from said patient and assigning to said patient a high risk of developing CAD if said SIRT1 level is below a predetermined threshold.
15. A method of determining acute coronary syndrome (ACS) in a patient, said method comprising quantifying the level of SIRT1 expression, particularly quantifying the level of SIRT1 protein, in a sample obtained from said patient, and using the obtained information during assessment of the probability of ACS, wherein it is indicative of a high probability of ACS if said SIRT1 level is below a predetermined threshold.
16. A method of determining ACS in a patient, said method comprising quantifying the level of SIRT1 expression, particularly quantifying the level of SIRT1 protein, in a sample obtained from said patient and assigning to said patient a high probability of ACS if said SIRT1 level is below a predetermined threshold.
17. The method according to any one of claims 12 to 16, wherein said method comprises contacting said sample obtained from said patient with an antibody specifically reactive to SIRT1.
18. The method according to claim 17, wherein said quantification of SIRT1 expression levels is performed by ELISA.
19. A method according to any one of claims 12 to 18, wherein SIRT1 isoform 1 is determined.
20. A kit for determining the risk of a patient to develop CAD, comprising an antibody against SIRT1.
21 . Use of an antibody against SIRT1 in the preparation of a kit for diagnosing ACS.
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