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WO2011018667A1 - Endonucléase g utilisée en tant que cible pour des maladies associées à l'hypertension et à l'hypertrophie - Google Patents

Endonucléase g utilisée en tant que cible pour des maladies associées à l'hypertension et à l'hypertrophie Download PDF

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WO2011018667A1
WO2011018667A1 PCT/GB2010/051338 GB2010051338W WO2011018667A1 WO 2011018667 A1 WO2011018667 A1 WO 2011018667A1 GB 2010051338 W GB2010051338 W GB 2010051338W WO 2011018667 A1 WO2011018667 A1 WO 2011018667A1
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endo
molecule
oligonucleotide
disease
endonuclease
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Stuart Cook
Christopher Mcdermott-Roe
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Medical Research Council
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/922—Ribonucleases (RNAses); Deoxyribonucleases (DNAses)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/321—Arterial hypertension

Definitions

  • the present invention relates to a method of identifying a molecule that modulates the 5 activity or expression of endonuclease G (Endo G). Moreover, the present invention provides a method for determining the level of endonuclease G activity and a method for treating or monitoring hypertension associated and/or hypertrophy associated disease.
  • Endonuclease G (Endo G), a mitochondrial endonuclease acting on both single- and double-stranded DNA, is the most abundant and active nuclease in all eukaryotic mitochondria. It is expressed ubiquitously in all eukaryotes and the gene is highly conserved in organisms ranging from Saccharomyces cerevisiae to humans.
  • Endo G plays a role in mitochondrial DNA replication, DNA recombination, and cell proliferation (Huang et al, 2006; Low, 2003). Subsequent studies have revealed the importance of Endo G in apoptotic DNA damage (Parrish et al, 2001, Wang et al, 2002). It was found that Endo G, located
  • Cardiovascular disease including stroke and heart failure, is the leading cause of death in the world. Cardiovascular disease can be caused by many conditions, and 30 diseases of the heart relate primarily to ischemia, hypertension and cardiac hypertrophy. Ischemic heart disease is characterised by reduced blood flow to the myocardium, usually as a result of atherosclerosis of the coronary arteries. Conditions, such as hypercholesterolaemia (high cholesterol levels) and diabetes, have been shown to increase the risk of ischemic heart disease. Endo G has been suggested to play a role in ischemic stress-induced heart dysfunction.
  • mitochondrial markers including Endo G
  • hypertensive heart disease is characterised by an abnormally high pressure on the wall of the blood vessels and hypertrophic heart disease is characterised by the enlargement of the heart due to thickening of the myocardium and can lead to heart failure and death.
  • some lifestyle factors, such as high salt diet and obesity have been linked to hypertension and hypertrophy, the underlying genetic causes of these conditions are less clear. Therefore, there is an urgent need to identify pharmaceutical agents for the treatment of hypertensive and hypertrophic heart diseases, and to develop a method for predicting the risk of developing these diseases and monitoring the progression thereof.
  • the present invention relates to the finding that Endo G plays an important role in hypertension and hypertrophy.
  • the first aspect of the invention provides a method for identifying a molecule suitable for the treatment of a disease selected from hypertension associated and hypertrophy associated disease, the method comprising contacting an Endo G protein with a candidate molecule and determining if said candidate molecule modulates the activity of said Endo G protein. It has been found that inhibition of Endo G activity reduces the severity of hypertension associated and hypertrophy associated diseases, such as, hypertension- and hypertrophy-induced contractile dysfunction in the heart and heart dilatation. In particular, it has been found that altering the expression level of EndoG affects the hypertrophic and remodelling response following addition of a hypertrophic agonist or an increase in blood pressure.
  • adenoviral-mediated overexpression of EndoG in isolated cardiacmyocytes followed by hypertrophic stimulation mitigates the expected increase in cell size.
  • EndoG-deficient mice develop exacerbated cardiac hypertrophy and more favourable contractile function when treated with a hypertension-inducing compound compared to wildtype mice. Accordingly, the inventors have determined that a molecule which modulates the activity of Endo G can be used for the treatment of a hypertension associated and/or a hypertrophy associated disease.
  • Endo G refers to a nuclear-encoded mitochondrial nuclease. Endo G is localised in the mitochondrion. It is widely distributed among animals and cleaves double-stranded DNA at (dG)n:(dC)n tracts, single-stranded DNA at consecutive C nucleotides and R loops (RNA-DNA hybrids). Endo G protein according to the present invention can be any mammalian Endo G protein. The sequences of mammalian Endo G proteins are well known in the art.
  • Endo G refers to a human Endo G protein, such as the protein described in Strausberg et al. , 2002 and Venter et al, 2001.
  • the terms "endonuclease G” and “Endo G” also cover its isoforms, which occur as a result of alternative splicing, and allelic variants thereof. For example, such isoforms in mouse have been described in Prats et al, 1997.
  • hypertension and “hypertrophy” are well known to those skilled in the art. Hypertension is defined as abnormally high blood pressure; and hypertrophy is defined as the increase in the volume of an organ or tissue due to the enlargement of its component cells.
  • the hypertension associated, and hypertrophy associated diseases are preferably hypertension-, and hypertrophy-induced diseases, respectively, i.e., the underlying cause of these diseases is hypertension, or hypertrophy.
  • the hypertension associated and hypertrophy associated diseases do not include diseases caused by ischemia. Hypertension associated diseases include, but are not limited to, hypertensive heart disease, hypertensive kidney disease and hypertensive vascular dysfunction.
  • Persistent hypertension leads to the thickening of walls of blood vessel and is one of the major contributory factors for congestive heart failure, cardiac arrhythmia (abnormal heart beat), hypertensive nephropathy (damage to kidney due to chronic high blood pressure) and dysfunction of skeletal muscle.
  • Hypertrophy associated diseases include, but are not limited to, hypertrophic cardiomyopathy, left ventricular hypertrophy, valvular disease, such as aortic stenosis, and skeletal muscle hypertrophy.
  • treatment refers to any process, action, application, therapy, or the like, wherein a mammal, including a human being, is subject to medical aid with the object of improving one's condition, directly or indirectly.
  • treatment also refers to prevention of hypertension associated or hypertrophy associated disease in which the occurrence of said disease is suppressed as compared with a suitable control, i.e., one not treated with the molecule identified by the present invention. Suppression means that said disease occurs at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% less than said control.
  • the candidate molecule for use in the method according to the first aspect of the present invention may be any suitable molecule.
  • the molecule is a small molecule, a peptide, an antibody, an aptamer or a mutant Endo G protein.
  • the small molecule can be any suitable small organic molecule or synthetic molecule. Such molecules can be obtained, for example, from combinatorial and natural product libraries.
  • the peptide includes any suitable peptide, including synthetic peptides selected from random peptide libraries, such as the tetrameric peptide libraries described in International patent application WO 01/85796, naturally occurring protein inhibitors for Endo G, such as Heat Shock Protein 70 (preferably human HSP70) and the Drosophila melanogaster CG4930 (Temme et ai, 2009), and any functional derivatives thereof.
  • the antibody of the present invention can be any antibody molecule that is specifically directed against Endo G.
  • the antibody may be polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library.
  • Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody.
  • the antibodies and fragments thereof may be humanised antibodies, for example, ones available from ProSci Inc. (Endo G Monoclonal Antibody No. PM-4583, PM-4577 and PM-4579).
  • antibodies with fully human variable regions (or their fragments) for example, ones available from ProSci Inc. (Endo G Antibody No. 3035), may also be used.
  • Antibodies may be produced by standard techniques, such as by immunisation, by using a phage display library or by recombinant DNA technology.
  • the antibody-binding fragments are commonly generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin or other proteases.
  • the aptamers are single- or double-stranded nucleic acids capable of specifically binding to selected targets and modulating the activity thereof. They can be generated by an in vitro selection process from pools of random oligonucleotide sequences or specifically designed.
  • a suitable method for generating an oligonucleotide from highly diverse pools of different nucleic acid molecules may be SELEX (systemic evolution of ligands by exponential enrichment), which has, for example, been described in Weiss et al, 1993, US Patent No. 5475096 and International Patent Application WO 91/19813.
  • the mutant Endo G proteins are Endo G proteins with altered sequences resulting in different characteristics and include dominant negative Endo G mutants and constitutively active Endo G mutants. Dominant negative mutants are functionally inactive forms of Endo G that are still capable of binding to the nucleic acid substrates of Endo G.
  • Said mutants can inhibit the activity of endogenous Endo G proteins by occupying the Endo G binding sites of the DNA substrates or other binding partners.
  • constitutively active mutants can increase the activity of endogenous Endo G proteins.
  • molecules of the present invention can be modified so as to aid the delivery of said molecules to the target site, e.g. mitochondria or the cytoplasm. Such modifications include, but are not limited to, providing other chemical or sugar groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, or modifying the molecules so that they are resistant to protease or nuclease cleavages.
  • modulates the activity means that the activity of Endo G is either increased or decreased. Preferably, the activity is increased or decreased by at least
  • the activity of Endo G that is modulated is the nuclease activity.
  • Methods for determining the nuclease activity are well known to those skilled in the art and are discussed in further detail below.
  • the molecule may bind to Endo G or its nucleic acid substrate to affect the nuclease activity.
  • the molecule binds to Endo G and affects the nuclease activity.
  • Any suitable method may be used to determining if the candidate molecule modulates the activity of Endo G. Suitable methods are well known to those skilled in the art. Preferably, the method according to the seventh aspect of the invention (see below) is used for this purpose.
  • Endo G activity reduces the severity of hypertension associated and hypertrophy associated diseases
  • molecules that reduce the activity of Endo G will be suitable for treating hypertension associated and hypertrophy associated diseases. Accordingly, it is preferred that the molecule that modulates the activity of Endo G inhibits the activity of Endo G.
  • a second aspect of the invention provides a method of identifying an agent suitable for the treatment of a disease selected from a hypertension associated and a hypertrophy associated disease, the method comprising contacting a candidate agent with an Endo G expression construct and determining if said agent modulates the level of expression from the expression construct. Since inhibition of Endo G activity reduces the severity of hypertension associated and hypertrophy associated diseases as mentioned above, it is determined that an agent that modulates the expression of Endo G can be used for the treatment of hypertension associated and/or hypertrophy associated disease.
  • the hypertension associated and hypertrophy associated diseases are as defined in the first aspect of the invention.
  • An Endo G expression construct is a DNA molecule comprising endogenous Endo G regulatory sequences, such as the promoter, and the coding sequence of a protein operably linked to the regulatory sequences.
  • the Endo G regulatory sequences are known to those skilled in the art and are generally located in the 2kb region 5' of the first exon of the Endo G gene.
  • the protein encoded by the expression construct can be any protein whose expression can be easily detected.
  • Said protein can be the Endo G protein or a reporter proteins such as ⁇ -galactosidase and luciferase.
  • the protein is Endo G.
  • Endo G expression construct may comprise the native nuclear DNA sequence that contains the Endo G regulatory sequences and the endogenous Endo G gene.
  • the candidate agent can be any molecule that either activates or inhibits Endo G gene expression, i.e. gene transcription or translation of the Endo G gene transcript.
  • the candidate agent is a small molecule, a peptide or a nucleotide.
  • the small molecule can affect the expression of Endo G by binding to either the regulatory sequences, such as the promoter, or transcriptional or translational machinery specific for the expression of Endo G. It can be a small organic molecule or synthetic molecule, for example, from combinatorial and natural product libraries.
  • the peptide can affect the expression of Endo G in the same manner as the small molecule described above.
  • the nucleotide can be any RNA or DNA molecule capable of inhibiting Endo G expression, for example, an antisense DNA or RNA, a small interfering RNA (siRNA), a microRNA (miRNA) or a ribozyme.
  • siRNA small interfering RNA
  • miRNA microRNA
  • ribozyme a small interfering RNA
  • the expression construct of the present invention must comprise the coding sequence of Endo G.
  • Antisense DNA and RNA molecules act to directly block the translation of the Endo G mRNA by binding specifically to the target mRNA.
  • siRNA, miRNA and ribozyme molecules can result in mRNA degradation, thereby inhibiting the translation of Endo G mRNA.
  • siRNA and miRNA for example, as described by Basnakian. 2006 and Huang el al., 2006, act by initiating the RNA interference process; ribozymes are enzymatic RNA molecules capable of catalysing the specific cleavage of RNA.
  • a third aspect of the invention provides the use of a molecule that modulates the activity of Endo G in the manufacture of a medicament for the treatment of a disease selected from a hypertension associated and a hypertrophy associated disease.
  • the third aspect of the present invention provides a method of treating a disease selected from a hypertension associated and a hypertrophy associated diseases, comprising administering to an individual in need of such treatment an effective dose of a molecule that modulates the activity of Endo G.
  • the terms used in respect of the third aspect of the present invention are as defined above.
  • the molecule that modulates the activity of Endo G can be a small molecule, a peptide, an aptamer, a mutant Endo G protein, an antibody or a nucleotide. Such molecules are as defined above with respect to the first aspect of the present invention.
  • the molecule that modulates the activity of Endo G is an antibody specifically directed against Endo G, Heat Shock Protein 70 (preferably human HSP70) and Drosophila melanogaster CG4930, and any functional derivatives thereof.
  • the molecule inhibits the activity of Endo G and is for the treatment of hypertension associated or hypertrophy associated diseases.
  • a fourth aspect of the invention provides the use of an agent that modulates the expression of Endo G in the manufacture of a medicament for the treatment of a disease selected from a hypertension associated and a hypertrophy associated disease.
  • the fourth aspect of the present invention provides a method of treating a disease selected from a hypertension associated and a hypertrophy associated diseases, comprising administering to an individual in need of such treatment an effective dose of an agent that modulates the expression of Endo G.
  • the agent that modulates the expression of Endo G can be a small molecule, a peptide or a nucleotide. Such agents are as defined above with respect to the second aspect of the present invention.
  • the agent inhibits Endo G expression and is for the treatment of hypertension associated or hypertrophy associated diseases. It is further preferred that the agent that inhibits the expression of Endo G is an antisense DNA or RNA, a small interfering RNA (siRNA), a microRNA (miRNA) or a ribozyme.
  • the molecule that modulates the activity of Endo G or the agent that modulates the expression of Endo G may be used in combination with one or more additional molecules conventionally used for the treatment of a hypertension associated or a hypertrophy associated disease.
  • Suitable conventional treatment molecules include diuretics, a beta blockers, angiotensin II receptor antagonists and calcium channel blockers.
  • a fifth aspect of the invention provides an oligonucleotide capable of being cleaved by an Endo G protein, wherein said oligonucleotide is labelled with a signal molecule and a quencher molecule, wherein the signal molecule and the quencher molecule are separated by a portion of said oligonucleotide having a high GC content and said portion comprises between 11 and 29 nucleotides.
  • the oligonucleotide of the present invention is particularly specific for Endo G.
  • the oligonucleotide of the present invention can therefore be used to determine the activity of Endo G with high specificity.
  • the portion of said oligonucleotide comprises between 12 and 27, more preferably between 13 and 25, still more preferably between 14 and 23, and even more preferably between 14 and 21 nucleotides.
  • a signal molecule is a molecule which generates a signal, wherein the signal can be detected easily and suppressed by the presence of a quencher molecule in close proximity.
  • the quencher molecule suppresses the signal by at least 80%, more preferably by at least 90% and most preferably by at least 99%.
  • the signal molecule is a fluorophore which emits fluorescence.
  • Fluorophores and quencher molecules that can be used in the present invention are well known to those skilled in the art.
  • fluorophores include 6-FAM, TET, HEX, Cy3, TMR, ROX, Texas red and LC red 640.
  • quenchers include BHQ-I, DDQ-I, QSY-21, Eclipse, Dabcyl and Iowa Black FQ.
  • a high GC content means that the collective number of guanine and cytosine nucleotides makes up at least 70%, preferably at least 80%, more preferably at least 90%, of the total number of nucleotides in said portion of the oligonucleotide.
  • the oligonucleotide is a double-stranded oligonucleotide having a portion with a high GC content comprising at least a sequence of (dG)n:(dC)n, wherein n can be any number between 1 and 12.
  • the oligonucleotide is a single-stranded oligonucleotide having a portion with a high GC content comprising a sequence of between 11 and 29, preferably between 12 and 27, more preferably between 13 and 25, still more preferably between 14 and 21 consecutive cytosine nucleotides. It is particularly preferred that said sequence contains 15 or 20 consecutive cytosine nucleotides.
  • said oligonucleotide is an R-loop.
  • a sixth aspect of the invention provides a method for determining the level of Endo G activity, the method comprising contacting an Endo G protein with an oligonucleotide according to the fifth aspect of the invention and detecting a signal. A high signal indicates a high level of Endo G activity and a low signal indicates a low level of Endo G activity.
  • Methods for detecting signals according to the present invention are well known to those skilled in the art. They include, but are not limited to, fluorescence spectroscopy and fluorescence detector as, for example, described in International patent application WO 2009/089543.
  • the terms "high” and “low” are relative terms as compared to a suitable control, which can be a defined level of Endo G protein activity.
  • the method according to the sixth aspect of the present invention is performed at about 37 ° C, i.e., between 3O 0 C and 40°C.
  • T he role of Endo G in hypertension and hypertrophy provides a way of using the level of Endo G activity to predict the risk of a hypertension associated or a hypertrophy associated disease and to monitor the progression thereof.
  • the present invention further provides a method of determining the risk of an individual developing a hypertension associated or a hypertrophy associated disease, or to monitor the progression of a hypertension associated or a hypertrophy associated disease comprising determining the level of Endo G activity in a biological sample obtained from the individual.
  • the biological sample may be any suitable sample such as a tissue sample. Suitable tissue samples include heart tissue, kidney tissue and muscle tissue.
  • the oligonucleotide according to the fifth aspect of the present invention is used to determine Endo G activity in the sample.
  • a seventh aspect of the invention provides a method of identifying a molecule that modulates the activity of Endo G, the method comprising contacting an Endo G protein with a candidate molecule and an oligonucleotide according to the fifth aspect of the invention and detecting a signal.
  • An increase in signal indicates that said molecule activates the activity of Endo G.
  • a decrease in signal indicates that said molecule inhibits the activity of Endo G.
  • the terms used in respect of the seventh aspect of the present invention are as defined above.
  • the method according to the seventh aspect of the present invention is performed at about 37°C, i.e., between 30 ° C and 40 ° C.
  • FIGURES Figure 1 is a Western blot which shows the expression of Endo G in various tissues and organs in SHR and BN rat.
  • Figure 2 is a picture of agarose gel electrophoresis demonstrating the nuclease activity of BN and SHR heart extracts.
  • Figure 3 shows the DNA and protein sequence analysis of Endo G in BN and SHR.
  • Figure 4 shows the effect of altering EndoG expression on the hypertrophic responsiveness of cardiacmyocytes.
  • Figure 5 shows the LV internal dimensions of wild-type (wt) and Endo G knockout (ko) mice during diastole and systole.
  • Figure 6 shows indexed LV mass in WT and KO mice with and without angll treatment as well as ventricular wall and internal chamber measurements.
  • Figure 7 shows myocyte cross sectional area in the hearts of WT and KO mice with and without angll treatment.
  • Figure 8 shows the negative correlation between human indexed LV mass and EndoG transcript abundance.
  • Figure 9 shows the effect of benzonase (a non-specific nuclease) on fluorescence generated by oligonucleotide substrates.
  • Figure 10 is a comparison of fluorescence generated by BN and SHR protein lysates using different oligonucleotides.
  • Figure 1 1 shows the effect of temperature on oligonucleotide cleavage by Endo G.
  • Figure 12 is a comparison of BN and SHR LV baseline nuclease activities using different amount of LV protein lysates.
  • Figure 13 is a comparison of LV baseline nuclease activities among different RI strains.
  • Figure 14 is a picture of agarose gel electrophoresis demonstrating the inhibitory effect of CG4930 (EndoGI) on Endo G nuclease activity
  • Figure 15 is the quantitative analysis of inhibition of Endo G nuclease activity by CG4930. DETAILED DESCRIPTION OF THE INVENTION
  • the spontaneously hypertensive rat is a widely studied rodent model of human hypertension. Despite being hypertensive, the SHR does not exhibit heart or kidney damage due to blood pressure effects. This is likely due to adaptive mutations that occurred during the selective breeding of the SHR. In the heart, these adaptive mutations might protect against the development of exuberant hypertrophy; preserve heart pumping function; and prevent apoptotic or necrotic myocardial cell damage. Analysis ofeQTL dataset
  • RNA samples from parental and BXH/HXB recombinant inbred (RI) strains and EndoG Transcript abundance was determined via a 2-step real time quantitative PCR (RT- QPCR).
  • cDNA was generated from LV total RNA using iScript (Bio-Rad) and then amplified with SYBR Green JumpStart Taq Ready Mix (Sigma) with gene-specific primers.
  • SYBR Green JumpStart Taq Ready Mix Sigma
  • Cj threshold cycle
  • CG4930 20 ⁇ g of BN-derived LV protein, 2 ⁇ g of the abovementioned oligonucleotide substrate and varying quantities of CG4930 (final concentration of 0-250 nM) were incubated in a 96-well plate in the plate-reader at 37 0 C for 2 hours, during which time fluorescence was read every 10 minutes as specified above.
  • Neonatal rat ventricular myocytes were maintained in serum-deficient media for 24 hours prior to addition of either an EndoG-encoding or an empty adenovirus (final concentration of 4 plaque forming units per cell). After a further 24 hours, phenylephrine (final concentration of 100 ⁇ M) and tritiated leucine (2 ⁇ Ci per culture) were added, after which cells were maintained in standard culture conditions for a further 24 hours. Cells were washed thoroughly with PBS before being lysed with 2 M sodium hydroxide and analysed for tritium incorporation.
  • mice C57BL6 wild-type and C 57BLo-EHdOG "7' mice were treated with isoproterenol (15 mg/kg/day) and angiotensin II (1.5 mg/kg/day) for 2 weeks via surgically-implanted osmotic minipumps, after which they were anaesthetised with isoflurane (4 %) and analysed by echocardiography using a trained genotype-blinded operator. Animals were subsequently sacrificed and organ weights were measured prior to snap freezing and storage.
  • Figure 1 shows that Endo G is absent in all tissues of SHR, but is expressed in LV, kidney and skeletal muscle of BN, with the highest expression level being in LV.
  • Immunoblotting revealed that whereas the left ventricle of the non-hypertrophic BN rat strain expresses EndoG, the SHR does not ( Figures Ia and Ib).
  • EndoG protein expression is highest in the heart and skeletal muscle whilst in the mouse, expression is again highest in the heart ( Figures Ia and Ic).
  • Analysis of neonatal-derived myocyte and non-myocyte populations revealed that EndoG is exclusively expressed by the myocyte fraction of the heart ( Figure Id).
  • Endo G is the only nuclease with significant activity in the heart.
  • isolated Sprague Dawley neonatal ventricular myocytes were infected with an EndoG-encoding adenovirus and subsequently treated with the beta adrenergic agonist, phenylephrine, to induce hypertrophy.
  • myocytes infected with a control adenovirus displayed an approximate and expected 30 % increase in protein synthesis, and those infected with the EndoG adenovirus failed to respond (Figure 4a).
  • MTT assays were carried out which demonstrated a negligible difference in health between control virus- and EndoG-infected cultures (Figure 4b).
  • FITC-conjugated WGA was used to stain and delineate the boundaries of individual myocytes in the heart, enabling calculation of cell size.
  • concentric hypertrophy is characterised at the cellular level by deposition of parallel-arranged sarcomere
  • cross sectional area was measured.
  • myocyte cross-sectional area from angll-treated wild-type mice was approximately 34 % higher than sham-operated wildtype mice whereas in angll-treated KO mice, it was approximately 64 % higher than sham-operated KO mice. This indicates that the augmented angll-induced LV mass increase exhibited by the KO mice is resultant from myocyte hypertrophy.
  • Oligonucleotides composed of 10, 15, 20 and 30 consecutive cytosine residues with a fluorophore (6-FAM) at the 5' end and a quencher molecule (BHQ-I) at the 3' end were synthesised by Eurogentec Ltd. When the oligonucleotide is intact, the fluorescence generated from 6-FAM (when excited) is quenched by the BHQ-I . However, when the oligonucleotide is cleaved (as occurs following incubation with nuclease enzymes, such as Endo G), the 6-FAM and BHQ- 1 diffuse away from one another, reducing quenching and leading to an increase in measurable fluorescence. Therefore, detection of fluorescence indicates oligonucleotide cleavage and the magnitude of the fluorescence indicates the extent of the cleavage and thus nuclease activity.
  • 6-FAM fluorophore
  • BHQ-I quencher molecule
  • the first goal was to determine whether cleavage of the oligonucleotide caused an increase in fluorescence.
  • Varying quantities of the oligonucleotide were incubated with a fixed quantity (5 units) of benzonase (a low specificity nuclease) for 30 minutes at 37 0 C.
  • a small volume of each reaction mixture was transferred to a single well in a 96-well plate and diluted 10-fold with water.
  • Incubating the oligonucleotide with benzonase caused a substantial increase in fluorescence (+ benzonase) compared to when the oligonucleotide was incubated with water alone (- benzonase), as shown in Figure 9.
  • the assay can be used to measure nuclease activity.
  • the next step is to determine whether this assay could be used to specifically measure Endo G activity.
  • protein solutions derived from the Endo G-containing BN and the Endo G-deficient SHR rats were incubated with a fixed quantity of the oligonucleotides. Following a 30 minute incubation as described above, fluorescence from each reaction was measured.
  • Figure 6 shows that the oligonucleotides composed of either 15 or 20 cytosine nucleotides generate a significant difference between the BN and the SHR protein lysates; whereas ones with 10 or 30 nucleotides do not.
  • Figure 13 shows that when fluorescence is measured for individual RI strains, the average signal generated by the BN strains was approximately 7-fold higher than that generated by the SHR strains.
  • CG4930 is a naturally occurring Endo G inhibitor found in Drosophila melanogaster (Tcmme et al, 2009).
  • Figure 14 shows the inhibitory effect of CG4930 (EndoGI), demonstrated by the intact plasmid DNA, on the nuclease activity of Endo G at concentrations greater than 0.15 ⁇ M.
  • EndoGI the inhibitory effect of CG4930
  • Figure 15 shows that the level of inhibition by CG4930 increases with the concentrations thereof, with 84% inhibition being achieved when 250 ⁇ M of CG4930 was used. This result suggests that the effects of molecules which modulate Endo G activity can be observed easily without the need for agarose gel electrophoresis. Moreover, the effects of these molecules can be quantified. Accordingly, this assay provides a potential screening method for identifying molecules that modulate Endo G activity.

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Abstract

La présente invention concerne un procédé d’identification d’une molécule qui module l’activité ou l’expression de l’endonucléase G pour le traitement d’une maladie choisie parmi une maladie associée à l’hypertension et une maladie associée à l’hypertrophie. La présente invention concerne également un procédé de détermination du niveau d’activité de l’endonucléase G et un procédé de traitement ou de suivi d’une maladie associée à l’hypertension et/ou d’une maladie associée à l’hypertrophie.
PCT/GB2010/051338 2009-08-14 2010-08-12 Endonucléase g utilisée en tant que cible pour des maladies associées à l'hypertension et à l'hypertrophie Ceased WO2011018667A1 (fr)

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US23402909P 2009-08-14 2009-08-14
US61/234,029 2009-08-14
GB0914318A GB0914318D0 (en) 2009-08-14 2009-08-14 Method
GB0914318.1 2009-08-14

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WO2011018667A1 true WO2011018667A1 (fr) 2011-02-17

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Cited By (1)

* Cited by examiner, † Cited by third party
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CN117929744A (zh) * 2023-12-14 2024-04-26 福建中医药大学 Rab31抑制剂在制备预防和/或治疗高血压的产品中的用途

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117929744A (zh) * 2023-12-14 2024-04-26 福建中医药大学 Rab31抑制剂在制备预防和/或治疗高血压的产品中的用途

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