Abstract:
A kit and a method for determining modulating agents of sirtuins; specifically, the modulation of sirtuin expression, by way of the detection and comparison of mRNAs in the sirtuins and β-actin. A process for modulating the activity of sirtuins, as well as pharmaceutical compounds and compositions capable of modulating the gene expression of sirtuins.

Description:
STATEMENT OF RELATED APPLICATIONS 
       [0001]    This patent application is based on and claims convention priority on Brazilian Patent Application No. PI0806044-4 having a filing date of 17 Oct. 2008. 
       BACKGROUND OF THE INVENTION 
       [0002]    1. Technical Field 
         [0003]    The invention herein describes a kit and a method for determining sirtuin modulating agents; specifically the modulation of the expression of sirtuins, by way of the detection and comparison of mRNAs of sirtuins and of β-actin. 
         [0004]    The invention herein also provides a process for the modulation of sirtuin activity as well as pharmaceutical compounds and compositions capable of modulating the gene expression of sirtuins. 
         [0005]    2. Related Art 
         [0006]    Sirtuins are part of a large group of enzymes, the histone deacetylases or HDACs family, whose principal role is to reverse the regulatory acetylation of histone-type proteins, by influencing directly in the structure of nucleosomes and, consequently, in gene transcription. Other than histones, a growing number of proteins have been identified as a target of HDACs, structural proteins and different transcription factors being among those. Sirtuins affect a broad number of physiological processes, including regulation of life expectancy, regulation of metabolic and enzymatic activity, cellular response to stress, neurodegeneration, DNA repair, rDNA recombination, apoptosis and the control of cellular proliferation. Currently, sirtuins are the important targets of research linked to caloric restriction, cancer, neurodegenerative diseases, muscular differentiation, inflammation and obesity. 
         [0007]    Different studies have been performed aiding in the identification of sirtuin modulators. Blander et al (“SIRT1 shows no substrate specificity in vitro” J Biol Chem. 280(11):9780-5, 2005) describe a previously unpublished method for identifying specificity for a sequence of deacetylase by using peptide libraries containing acetylated lysine. After incubation with SIRT1, the subassembly of deacetylated peptides was captured selectively using a biotinylated N-hydroxysuccinimide photolabile linker and small beads of streptavidin, subsequently analysed. Said studies revealed that the recognition of the substrate by SIRT1 occurs irrespectively of the sequence of amino acids close to the acetylated lysine. Furthermore, Garske et al. (“SIRTI top 40 hits: use of one-bead, one-compound peptide libraries and quantum dots to probe deacetylase specificity.” Biochemistry 45(I):94-101, 2006) also describe an new method of high scalability for determining the specificity of deacetylase substrates using an acetylated peptide library of one-bead, one-compound (OBOC). A library of 104,907 unique peptides was constructed and screened utilising NAD+− dependent deacetylase SIRT1 for more efficient peptide sequences. As a result, it was discovered that SIRT1 can discriminate between peptide substrates depending on the context in which they find themselves. 
         [0008]    The document, WO 08/27379, describes a method for monitoring the modulation of sirtuin activity by determining the level of acetylation of sirtuin substrates. 
         [0009]    Documents, WO 07/149270 and WO 06/096780, describe a method for screening modulating molecules of sirtuin activity, which include as a crucial stage the determination of the level of acetylation of a specific peptide. 
         [0010]    The document, WO 07/084162, describes new molecules with sirtuin inhibiting activity that are useful in the treatment and/or prevention of cancer and autoimmune diseases. 
         [0011]    The abovementioned studies illustrate the difficulties and discrepancies that arise from methods for identifying specific substrates for sirtuins. With regard to peptide libraries, such libraries were limited to small sequences and are influenced by amino acids that can be incorporated and they provide information about artificial sequences in an artificial context. 
         [0012]    As the methods proposed by publicly disclosed research do not demonstrate a consensus regarding their results, one can see that there exists a need for a method for identifying sirtuin modulators and in an appropriate cellular environment. The identification of modulating agents will help to clarify the role of sirtuins in cells and will aid in the development of diagnostic tests and the treatment of patients with modulators of the respective activity, as well as methods for identifying new modulating compounds. 
         [0013]    The invention herein is different from all the documents relating to publicly disclosed research, as it proposes a new method for determining the modulation of sirtuin activity through the modulation of its gene expression. None of the documents from publicly disclosed research describes or even suggests that sirtuin activity can be modulated with agents that modulates its expression. Until now, publicly disclosed research has been concerned solely with the modulation of enzymatic sirtuin activity, which has already been expressed in the cell. 
       BRIEF SUMMARY OF THE INVENTION 
       [0014]    Firstly, the invention herein provides a method and a kit for determining modulating agents of sirtuin gene expression. 
         [0015]    An object of this invention is therefore a method for determining modulating agents comprised of the following stages:
       a) contacting tissue cell(s) comprising at least of gene of the sirtuin family and the β-actin gene, with a modulating agent;   b) determining the relative abundance of mRNA of gene(s) of the sirtuin family;   c) determining the relative abundance of mRNA of the β-actin gene; and   d) determining the mRNA SIRTImRNA β-actin ratio.       
 
         [0020]    In particular, tissue cell(s) comprising the abovementioned genes are those of zebrafish. 
         [0021]    A further object of the invention herein is a kit for determining modulating agents comprising:
       a) means for determining the relative abundance of mRNA of sirtuin-family genes; and   b) means for determining the relative abundance of mRNA of the β-actin gene.       
 
         [0024]    Secondly, the invention herein provides a process for modulating sirtuins, in which the modulation of sirtuins is performed by way of the modulation of the gene expression of sirtuins. 
         [0025]    An additional object of the invention herein is a modulating agent of sirtuin expression. 
         [0026]    Another object of the invention herein is a pharmaceutical composition comprising:
       a) a modulating agent of sirtuin expression; and   b) an acceptable pharmaceutical vehicle.       
 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWING FIGURES 
         [0029]    Further features of the invention can be gathered from the accompanying drawings, in which: 
           [0030]      FIG. 1  demonstrates one of the trees generated that was constructed utilising a proportional distance (p-distance) by way of the Neighbour-Joining (NJ) method, using the MEGA4.02 program. The tree contains seven well-settled terminal cladistic groupings with a high support amount, corresponding to each one of the sirtuins described. The phylogenetic tree consistently grouped together the orthological sequences, SIRT1-SIRT7 of  Danio rerio  (Dr),  Gallus gallus  (Gg),  Mus musculus  (Mm) and  Homo sapiens  (Hs) 
           [0031]      FIG. 2  demonstrates the pattern of expression of SIRT1 (A), SIRT2 (B), SIRT3 (C), SIRT3.2 (D), SIRT4 (E), SIRT5 (F), SIRT6 (G) and SIRT7 (H) in zebrafish. These results are expressed as an mRNA ratio of sirtuin/β-actin (mean ±S.E.) from seven repeated, independent experiments. On the Y axis, I is the rate of mRNA, on the X axis, 1 is the spleen, 2 is the gills, 3 is the brain, 4 is the heart, 5 is the liver, 6 is the female sexual organ, 7 is the male sexual organ, 8 is muscle and 9 is the kidney, I is β-actin, II is SIRT1, Ill is SIRT2, IV is SIRT3, V is SIRT3.2, VI is SIRT4, VII is SIRT5, VIII is SIRT6, IX is SIRT7. 
           [0032]      FIG. 3  demonstrates the evaluation of the effect of resveratrol in the modulation of sirtuin activity in zebrafish in different tissues in 4 animals (groups 1 to 4 on the X axis). On the Y axis, I is the rate of mRNA X, a is muscle and b is liver. 
       
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0033]    The examples shown here have the single aim of exemplifying one of the numerous ways of carrying out the invention, however, without detracting from its purpose. 
         [0034]    Sirtuins 
         [0035]    For the purpose of this invention, the expression, “sirtuins”, refers to the sequences (DNA and/or RNA), as well as the respective proteins, comprising at least 50% of homology with the sequences (DNA and/or RNA), as well as the respective proteins of the chosen genes of the group comprising SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT5, SIRT6, SIRT7 and combinations of the same. 
         [0036]    The respective sequences of the SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6 and SIRT7 genes are described in the GenBank, under the access codes for the species,  Danio rerio  (XP — 001334440, AAH67165, XP — 684225, XP — 690925 (SIRT 3.2), AAH83418, AAH75987, NP — 001002071, XP — 698800),  Homo sapiens  (Q96EB6, Q8IXJ6, Q9NTG7, Q9Y6E7, Q9NXA8, Q8N6T7, Q9NRC8),  Mus musculus  (BAD38898, BAS38897, XP — 420920, XP — 415273, XP — 418925, NP — 001034409, NP — 001026277) and  Gallus gallus  (NP — 062786, NP — 071877, CAJ18608, Q8R216, NP — 849179, NP — 853617, AAP83960). 
         [0037]    Ideally, sirtuins that are useful in the invention herein possess DNA sequences with at least 50% homology with the sequences illustrated in  FIG. 1 . 
         [0038]    β-Actin 
         [0039]    For the purposes of this invention, the expression, “β-actin” refers to sequences (DNA and/or RNA), as well as respective proteins, comprising at least 50% homology with the sequences (DNA and/or RNA), as well as respective proteins of the β-actin gene. 
         [0040]    Ideally, the β-actin sequence is described in the GenBank under the access number, NM — 001101. 
         [0041]    Cells comprising at least of gene of the sirtuin family 
         [0042]    For the purposes of this invention, adequate cells are cells of vertebrate animals. Preferably, the vertebrate should be chosen from the group comprising fish, birds, mammals and combinations of the same. 
         [0043]    Ideally, the vertebrate should be chosen from the group comprising  Danio rerio, Gallus gallus, Mus musculus, Homo sapiens  and combinations of the same. 
         [0044]    The cell is chosen from different tissues in the animal. Examples of organs/tissues include, but are not limited to, the brain, kidney, sexual organs (male and female), spleen, gills, liver, muscle, heart and combinations of the same. 
         [0045]    The zebrafish ( Danio rerio ) possess numerous advantageous characteristics that firstly make it the principal vertebrate model for biology studies in development and genetics and, subsequently, expansion to other areas of biological sciences. The zebrafish combines the characteristic of being a vertebrate with the study size of an invertebrate organism. The knowledge accumulated from the “Zebrafish Genome Project” together with the capability to quickly absorb chemical substances directly from water have the effect that it is increasingly used as well as a model species for studies in toxicology, pharmacology and human diseases. 
         [0046]    The identification of genes related to sirtuins in zebrafish genomes and the determination of expression patterns in different tissues constitute a very powerful tool for screening modulating molecules for the activities of these proteins. 
         [0047]    Method for Determining Modulating Agents 
         [0048]    The method for determining modulating agents of sirtuin gene expression is comprised of the following stages:
       a) contacting cells comprising at least on gene of the sirtuin family and the β-actin gene, with a modulating agent;   b) determining the relative abundance of mRNA of the sirtuin-family gene;   c) determining the relative abundance of the mRNA of the β-actin gene; and   d) determining the mRNA SIRTImRNA β-actin ratio.       
 
         [0053]    Ideally, one determines the relative abundance by way of mRNA amplification tests in both genes. Said tests are common knowledge to experts in the subject and do not need to be described here in greater detail. 
         [0054]    In the event that the compound possesses a mRNA SIRTImRNA β-actin ratio that is greater than 1, the compound is classified as a positive modulator (amplifier) of sirtuin gene expression. 
         [0055]    In the event that the compound possesses a mRNA SIRTImRNA β-actin ratio that is less than 1, the compound is classified as a negative modulator (inhibitor) of sirtuin gene expression. 
         [0056]    Pharmaceutical Composition 
         [0057]    For the purposes of this invention, pharmaceutical compositions include all compositions that contain an active principle, with prophylactic, palliative and/or curative purposes, which acts so as to maintain and/or restore homeostasis. It can be administered topically, parenterally, enterally and/or intrathecally. 
         [0058]    The expression, “pharmaceutically acceptable”, is employed here when referring to compounds, materials, compositions and/or dosage methods which, within the medical field, are appropriate for use in contact with human and animal tissue without excessive toxicity, irritation, allergic response or other problems or complications, it having a reasonable benefit/risk relationship. 
         [0059]    The composition in the invention herein can be administered by way of an oral dosage, in the form of tablets, capsules (each one includes sustained liberation or formulations with liberation time), pills, powders, granules, elixirs, dyes, suspensions, syrups and emulsions. 
         [0060]    The pharmaceutical composition in this invention is comprised of:
       a) a modulating agent for sirtuin expression; and   b) a vehicle which is acceptable pharmaceutically,
 
in which the mRNA SIRTImRNA β-actin ratio of the cell without the presence of the modulating agent is different from the mRNA SIRTImRNA β-actin ratio of the cell without the presence of the modulating agent.
       
 
       Example 
       [0063]    Eight genes related to sirtuins were identified in the zebrafish genome SIRT1-7 and a SIRT3 paralogue, named S1RT3.2 (Table 1) utilising the sequences deduced from  Homo sapiens  amino acids (Q96EB6, Q8IXJ6, Q9NTG7, Q9Y6E7, Q9NXA8, Q8N6T7, Q9NRC8)  Mus musculus  (BAD38898, BAD38897, Xp — 420920, XP — 415273, XP — 418925, NP — 001034409, NP — 001026277) and  Gallus gallus  (NP — 062786, NP — 071877, CAJ18608, Q8R216, NP — 849179, NP — 853617, AAP83960). The identity of each one of the eight sequences of zebrafish was confirmed by way of phylogenetic analysis ( FIG. 1 ) 
         [0000]    
       
         
               
               
             
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Sequences Summary: Access number, primers sequence and PCR 
                   
               
               
                 amplification conditions. 
               
             
          
           
               
                   
                 PCR Conditions 
               
             
          
           
               
                   
                   
                   
                 Tm 
                   
                   
               
               
                 Sirtuin 
                 GenBank ID 
                 Primers (5′-3′) 
                 (° C.) 
                 Ciclos 
               
               
                   
               
               
                 SIRT 1 
                 XP_001334440 
                 F-CAGCTCTGCTACAATTCATCGCGTC 
                 62 
                 30 
                   
               
               
                   
                   
                 R-AATCTCTGTAGAGTCCAGCGCGTGTG 
               
               
                   
               
               
                 SIRT 2 
                 AAH67165.1 
                 F-TCTCTGAAGAAATTCCTAAGTGCGATTCC 
                 61 
                 30 
               
               
                   
                   
                 R-TTATCTGAATCAAAATCCATTCCGCCTC 
               
               
                   
               
               
                 SIRT 3 
                 NP_001073643 
                 F-CATTAAATGTGGTGGAACAAGAGGCCTG 
                 61 
                 30 
               
               
                   
                   
                 R-AGTTCCTCTCCTTTGTAATCCCTCCGAC 
               
               
                   
               
               
                 SIRT 3.2 
                 NP_001038173.1 
                 F-CGGCAGGCTGATGAAGCTTGGTCG 
                 63 
                 30 
               
               
                   
                   
                 R-TAGCTTGCTTGGCTTCCTCTGCAGG 
               
               
                   
               
               
                 SIRT 4 
                 AAH83418.1 
                 F-TGTGGTGAACTGACTCCTCGTGCTGAGC 
                 63 
                 30 
               
               
                   
                   
                 R-CGGAAGTTTTCTTTCACTAGCAGCGAGG 
               
               
                   
               
               
                 SIRT 5 
                 AAH75987.1 
                 F-CCACGGTAGTCTGTTTAAAACCCGCTG 
                 61 
                 30 
               
               
                   
                   
                 R-AGTGATATTTGAAGCGTTGGGTAGCAGG 
               
               
                   
               
               
                 SIRT 6 
                 NP_001002071 
                 F-GGACTGGGAGGACTCTCTGCCCGAC 
                 68 
                 30 
               
               
                   
                   
                 R-GCCCGGCCCACTCCGGAACG 
               
               
                   
               
               
                 SIRT 7 
                 AA155852.1 
                 F-GCATTTTGGAGAACGTGGCACTTTGG 
                 61 
                 45 
               
               
                   
                   
                 R-GTTTAGCCATGCTGAAGATGGGGTCC 
               
               
                   
               
             
          
         
       
     
         [0064]    The mapping of the pattern and levels of expression in each one of the eight genes related to zebrafish sirtuin was determined in eight tissues (spleen, gills, brain, heart, liver, female sexual organs and male sexual organs, skeletal muscle and kidneys), by way of the analysis of semi-quantitative RT-PCR comparing the relative abundance of mRNA of each one of the genes related to sirtuins with the mRNA of the gene codifying for β-actin (mean ±E.P.) ( FIG. 2  and Table 2). 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Gene expression pattern: relative expression of mRNA in genes related 
               
               
                 to sirtuin in different zebrafish tissues. 
               
             
          
           
               
                   
                 Optical Density (O.D.) for SIRTIβ-actin mRNA ratio (mean ± S.E) 
               
             
          
           
               
                 Tissues 
                 SIRT1 
                 SIRT2 
                 SIRT3 
                 SIRT3.2 
                 SIRT4 
                 SIRT5 
                 SIRT6 
                 SIRT7 
               
               
                   
               
               
                 Spleen 
                 1.07 ± 0.07 
                 * 
                 0.61 ± 0.05 
                 * 
                 * 
                 0.44 ± 0.05 
                 0.47 ± 0.03 g   
                 0.56 ± 0.04 d,g   
               
               
                 Gills 
                 1.01 ± 0.01 
                 0.34 ± 0.00 a,d,e   
                 0.57 ± 0.16 
                 * 
                 0.36 ± 0.10 f   
                 0.24 ± 0.01 c,f   
                 0.38 ± 0.01 g   
                 * 
               
               
                 Brain 
                 0.98 ± 0.05 
                 0.65 ± 0.08 b   
                 0.52 ± 0.04 
                 0.37 ± 0.03 
                 0.46 ± 0.03 
                 0.55 ± 0.03 
                 0.56 ± 0.04 g,h   
                 0.79 ± 0.10 g   
               
               
                 Heart 
                 1.05 ± 0.04 
                 0.77 ± 0.05 b,h   
                 0.46 ± 0.05 
                 * 
                 0.61 ± 0.07 h   
                 0.45 ± 0.09 
                 0.53 ± 0.06 g,h   
                 0.83 ± 0.05 
               
               
                 Liver 
                 1.33 ± 0.34 
                 0.56 ± 0.02 
                 0.41 ± 0.04 
                 0.49 ± 0.09 
                 0.33 ± 0.06 f   
                 0.56 ± 0.12 
                 0.98 ± 0.17 a,b,c.d,e,f,h   
                 1.19 ± 0.04 a,c,f,i   
               
               
                 Female 
                 1.00 ± 0.04 
                 0.51 ± 0.06 
                 0.43 ± 0.03 
                 0.54 ± 0.07 
                 0.39 ± 0.06 
                 0.61 ± 0.05 b   
                 0.59 ± 0.17 g,h   
                 0.79 ± 0.05 g   
               
               
                 Sexual Organ 
               
               
                 Male 
                 1.97 ± 0.49 h,i   
                 0.68 ± 0.09 b   
                 0.64 ± 0.17 
                 0.51 ± 0.08 
                 0.52 ± 0.12 h   
                 0.56 ± 0.01 
                 0.65 ± 0.03 g,h   
                 0.92 ± 0.11 f,i   
               
               
                 Sexual Organ 
               
               
                 Skeletal 
                 0.87 ± 0.01 g   
                 0.37 ± 0.01 e   
                 0.38 ± 0.05 
                 * 
                 0.19 ± 0.01 e,d,f   
                 0.29 ± 0.02 f   
                 0.19 ± 0.00 a,c.d,e,f   
                 * 
               
               
                 Muscle 
               
               
                 Kidney 
                 0.89 ± 0.04 g   
                 * 
                 0.64 ± 0.06 
                 * 
                 0.69 ± 0.03 b,g,m   
                 0.62 ± 0.09 b,h   
                 0.41 ± 0.02 g   
                 0.45 ± 0.09 d,g   
               
               
                   
               
               
                 The results were analysed by ANOVA followed by the post-hoc Tukey HSD test, considering P ≦ 0.05 as significant. 
               
               
                 The mRNA levels were significantly different from 
               
               
                   a spleen, 
               
               
                   b gills, 
               
               
                   c brain, 
               
               
                   d heart, 
               
               
                   e liver, 
               
               
                   f female sexual organ, 
               
               
                   g male masculine organ, 
               
               
                   h skeletal muscle, 
               
               
                   i kidney. 
               
             
          
         
       
     
         [0065]    So as to evaluate modulation in this protein family, the effect of resveratrol (3.4′,5-trihydroxy-trans-stilbene), a phytoalexin which is found is some food items, such as egg shell, peanuts and red wine, was evaluated and it promoted a significant increase in SIRT1 gene expression in skeletal muscle and liver in the proposed model ( FIG. 3 ).