Patent Application
20070016970
This application claims the benefit of European Application No. 05106404.6, filed Jul. 13, 2005, which is hereby incorporated by reference in its entirety.
The present invention relates to guinea pig chymase, the nucleotide sequence that encodes the guinea pig chymase, and methods for identifying compounds that inhibit the activity of chymase and PAOD (peripheral arterial occlusive disease) and counteract asthma, based on the guinea pig chymase.
Chymase is a chymotrypsin-like serine protease which, with the exception of the rat, occurs in the secretory granules of mast cells. In immunology, mast cells are known above all for their role in allergic and fibrotic reactions. If the mast cells are activated, then chymase is released together with histamine and many other mediators via degranulation into the extracellular space. After its release, chymase develops a range of important inflammation-promoting effects. Chymase splits various physiologically and pathologically important proteins, such as e.g. the stem cell factor, big endothelin, TGF beta or apolipoproteins, and is also capable of activating collagenase proteolytically. In addition to these effects, it is assumed that one of the main tasks of human chymase is the production of angiotensin II from angiotensin I. For this reason, it is also speculated that chymase plays an important role in the case of heart failure, high blood pressure, allergy, dermatitis, rheumatic arthritis, asthma and chronic inflammations.
Species such as humans have only one chymase gene, whereas other species have several. It appears as though chymases are very similar to one another if only one is contained in an organism. If several are present, then there appears to be a division of tasks and adaptation to these tasks. Thus animal models with just one chymase are the most suitable for preclinical tests, as humans likewise only have one chymase. In the case of rodents in particular, such as mice and rats, we find numerous chymase genes, and even in hamsters there are two chymase genes.
Therefore, there is a need to find a species of animal that has only a single chymase isoform which at the same time is highly homologous to human chymase, for use in preclinical tests.
The present invention accordingly relates to a nucleic acid sequence comprising the sequence of Seq ID No. 1 (
Since the guinea pig is the most suitable animal model with a single chymase bioform, the present invention also relates to a guinea pig with somatic cells and germ cells with a mutated chymase gene, wherein at least one allele is functionally interrupted through homologous recombination of the mutated chymase gene, wherein this functional interruption inhibits the expression of the functional chymase gene in the guinea pig cells.
The present invention thus also relates to the following methods for identifying modulators of chymase activity a method for screening compounds that modulate the activity of chymase, comprising peptides: a) bringing a compound into contact with isolated polypeptide sequence of SEQ ID NO. 2; and b) determining the activity of guinea pig chymase; wherein a compound that inhibits or stimulates the activity of the guinea pig chymase is a compound that modulates the activity of the guinea pig chymase.
The present invention also provides a method for identifying compounds that modulate the activity of chymase, comprising a) bringing a compound into contact with the host cell according to claim 5; and b) determining the activity of the guinea pig chymase that is expressed in the host cell; wherein a compound that inhibits or stimulates the activity of the guinea pig chymase is a compound that modulates the activity of the guinea pig chymase.
The present invention also provides a method for identifying compounds that modulate the activity of chymase, comprising a) administering a compound to guinea pigs; and b) determining the activity of the guinea pig chymase; wherein a compound that inhibits or stimulates the activity of the guinea pig chymase is a compound that modulates the activity of the guinea pig chymase.
The present invention finds a species with only a single chymase isoform which is highly homologous to human chymase through the cloning and provision of guinea pig chymase. For this, both genomic and total RNA were isolated from guinea pig organ samples, in particular from the heart. In order to be able to duplicate chymase or chymase-like sequences in a targeted manner, it was necessary to develop primers with a specificity in respect of these sequences. The primers that were created here are listed in Table 5. With these primers, PCR amplifications starting out from genomic DNA were carried out. In order to enable a statement to be made about the results of the PCRS, the sequences of the plasmid inserts had to be determined. Via a database search, these were then compared with all DNA sequences known to date, and examined for homologies. It was also possible thus to convert the DNA sequences into an amino acid sequence of the resulting protein.
The sequencing of 11 samples yielded the DNA sequences of serine proteases that are already known and are very similar to chymase. However for one sample, for the first time a chymase-like guinea pig part-sequence was found which is shown in
The batches that yielded results contained the primers GP_CHY—12_F and GP_CHY—17_R, and the primers GP_CHY—15_F and GP_CHY—17_R. It would thus appear that of all the primers developed so far, the primer GP_CHY—17_R has the highest specificity in relation to chymase-like sequences in guinea pigs.
In a next step, on the basis of the sequence isolated from the genomic DNA, the encoded sequence was determined, in that a further fragment of the guinea pig chymase was amplified using specific primers (table 6) from guinea pig cDNA as a template. Starting out from this part-sequence, further primers were selected (table 7), in order to determine a further cDNA sequence of guinea pig chymase. In various reactions, it was possible to determine a further sequence, which is shown in
Altogether, 437 sequencings were carried out. In the course of this, guinea pig chymase was found 7 times, granzyme 7 times, but no alternative chymases were found.
The invention accordingly relates to a nucleic acid sequence comprising the sequence of Seq ID No. 1 (
The guinea pig chymase polypeptide sequence was compared with known sequences (tables 1 and 2).
Of the animal species that are compared here, the guinea pig is the one with the highest identity and homology to human chymase (77.9 and 86.3%). The chymases of human, mouse and guinea pig are so similar to one another that all these chymases are α chymases.
Since the guinea pig is the most suitable animal model, the present invention relates to a guinea pig with somatic cells and germ cells with a mutated chymase gene, wherein at least one allele is functionally interrupted through homologous recombination of the mutated chymase gene, wherein this functional interruption inhibits the expression of the functional chymase gene in the guinea pig cells.
The present invention thus also relates to the following methods for identifying modulators of chymase activity and inhibitors of PAOD:
A method is disclosed for screening compounds that modulate the activity of chymase, comprising the steps: a) bringing a compound into contact with isolated guinea pig chymase polypeptide according to claim 2; and b) determining the activity of guinea pig chymase; wherein a compound that inhibits or stimulates the activity of the guinea pig chymase is a compound that modulates the activity of the guinea pig chymase.
Also disclosed is a method for identifying compounds that modulate the activity of chymase, comprising the steps: a) bringing a compound into contact with isolated polypeptide according to claim 2; and b) determining the activity of guinea pig chymase; wherein a compound that inhibits or stimulates the activity of the guinea pig chymase is a compound that modulates the activity of the guinea pig chymase.
A further method is a method for identifying compounds that modulate the activity of chymase, comprising the steps: a) bringing a compound into contact with the host cell according to claim 5; and b) determining the activity of the guinea pig chymase that is expressed in the host cell; wherein a compound that inhibits or stimulates the activity of the guinea pig chymase is a compound that modulates the activity of the guinea pig chymase.
The present invention also discloses a method for identifying compounds that modulate the activity of chymase, comprising the steps: a) administering a compound to guinea pigs; and b) determining the activity of the guinea pig chymase; wherein a compound that inhibits or stimulates the activity of the guinea pig chymase is a compound that modulates the activity of the guinea pig chymase.
Finally, the present invention relates to a method for identifying compounds that inhibit PAOD, comprising the steps: a) administering to guinea pigs a compound that inhibits the guinea pig chymase; b) causing PAOD, and c) determining the extent of PAOD; wherein a compound that inhibits PAOD is a compound that leads to a reduction in the extent of the PAOD that is caused by step b). Preferably, PAOD is caused by administering lauric acid, through damage to a vessel with a balloon catheter or by feeding a diet with a high proportion of fat. Causing PAOD by administering lauric acid is particularly preferred. Another means of causing PAOD that is especially preferred is through damage to a vessel of the guinea pig with a balloon catheter, wherein the vessel is preferably the carotid.
The extent of PAOD is determined using familiar methods. For example, the extent of PAOD is determined by determining the formation of a neointima following a balloon catheter injury e.g. in the carotid of guinea pigs. Another possibility is determining the fatigue of guinea pigs running on a treadmill in the lauric acid model (Kawamura et al., 1985, Arzneimittelforschung 35/7A: 1154-1156).
The present invention also relates to a method for identifying compounds to combat asthma, comprising the steps: a) administering to guinea pigs a compound that inhibits the guinea pig chymase; b) causing asthma; and c) determining the extent of asthma, wherein a compound that inhibits asthma is a compound that leads to a reduction in the extent of the asthma that is caused by step b). In this method, familiar methods are used for inducing asthma and for determining the extent of asthma.
The term “compound”, as used herein, refers to and encompasses small molecules, and specifically and preferably, small molecules analogous to the compounds listed in Table 8. More preferably, the term compound refers to MSK SF2809-V: N-(2-{2-[(4-Hydroxy-1-methyl-2-oxo-1,2-dihydro-quinolin-3-yl)-phenyl-methyl]-1H-indol-3-yl}-ethyl)-acetamide and Toa Eiyo TY51076: 4-(5-Fluoro-3-methyl-benzothiophene-2-sulfonylamino)-3-methanesulfonyl-benzoic acid methyl ester.
The present invention also relates to sequences, methods and guinea pigs as described above, in particular in relation to the following examples.
The following examples are provided for illustrating purposes and are not intended to limit the scope of applicants' invention.
In order to be able to demonstrate the existence and activity of chymase in guinea pigs, total RNA was isolated. The heart of a guinea pig was used as the organ sample.
Disintegration of the cells took place through the use of so-called Lysing Matrix D Column. For this, 5×10 mg and 5×30 mg of the heart of a guinea pig were filled respectively into tubes with small ceramic balls and overlaid with 300 μl of a mixture of RLT buffer (RNeasy kit) and β-mercaptoethanol (10 μl β-mercaptoethanol per 1 ml of RLT buffer). Subsequently, the tubes were shaken for 2×20 seconds with a FastPrep homogeniser, through which disintegration of the cells was effected through shearing forces. Through centrifugation, the coarsest cell debris was sedimented and the supernatant fluid was transferred into clean tubes.
Subsequently, 590 μl of distilled water and 10 μl 1 mg/ml protein kinase K were added to each tube, the solutions were mixed through inverting and incubated for 10 minutes at 55° C. to break down the proteins. Through centrifugation for 3 minutes at 10,000 rpm and at room temperature, the coarsest cell debris was sedimented and 900 μl of the supernatant fluid was transferred into clean tubes.
The isolation of the total RNA from this mixture of proteins and other cell components was carried out using the RNeasy Mini Kit in accordance with the instructions (see Manual RNeasy Kit, protocol for “Isolation of Total RNA from Heart, Muscle and Skin Tissue”).
The concentrations of the various resulting RNA solutions were subsequently determined photometrically, at a wavelength of 260 nm.
The production of cDNA was carried out by means of the Transcriptor First Strand cDNA Synthesis Kit according to the instructions, as described below. Here, two different batches were prepared, for the use of two different primers.
To remove low-molecular fragments and unused constituents of the RT-PCR, the different batches were purified by means of the QIAquick PCR Purification Kit according to the instructions for microcentrifuges, as follows:
Another possibility for demonstrating chymase in guinea pigs was the use of genome DNA. This method was used since it could not be assumed with certainty that the gene that was sought was in fact expressed.
To obtain genome DNA, as in the case of RNA, disintegration of the cells was necessary. Once again, this was carried out via the aforementioned use of Lysing Matrix D columns. For this, 2×100 of the heart of a guinea pig were filled respectively into tubes with small ceramic balls and overlaid with 1.2 μl of digestion buffer. The composition of this buffer is shown in table 2-13.
Subsequently, the tubes were shaken for 2×20 seconds with a FastPrep homogeniser, through which disintegration of the cells was effected through shearing forces.
The separation of proteins and DNA took place via phenol extraction by means of Phase Lock Gel according to the instructions, as follows.
Subsequently, the DNA was cleansed and concentrated via ethanol precipitation, as detailed below.
In order to be able to identify chymase sequences where the respective gene is inactive, it is necessary to multiply these sequences from genome DNA templates by means of a PCR. In this part-experiment, different degenerated primers which have a specificity for guinea pig chymase were developed and used for this purpose.
It was presumed that chymase showed great homology in all animal models. Thus starting from the sequence for hamster chymase 1, which was already known, a database search was started for other known DNA sequences with as much in common as possible. The positive sequences of this search, which included e.g. many serine proteases and chymases of mice or rats, were then compared with one another, and from the regions with the widest variations between the individual sequences, primers were developed of which it could be assumed that they would bind chymase or chymase-like sequences. Within the individual primers, to some extent variations were allowed, in order to obtain a greater selection of sequences. These variations were produced by allowing several different bases at individual positions of the sequences. Such primers are termed degenerated. The variations were expressed via the generally applicable letter codes shown in table 4; production of these primers was undertaken by a contract firm.
Through the use of the degenerated primers, the attempt was to be made to amplify chymase or chymase-like sequences. For this purpose, each of the F primers was combined with each R primer.
The PCR batches were composed as follows:
The PCR proceeded according to the following program:
The PCR products were analysed with a 1% agarose/EtBr gel.
In order to be have sufficient material in respect of PCR products that have been obtained available for further investigations, these were amplified by means of transformation and via the cultivation of bacterial cultures.
In order to remove low-molecular fragments and other interference substances for the later transformation, selected PCR batches were purified with the QIAquick PCR Purification Kit according to the instructions, as described above.
Since several products or low-molecular interference fragments frequently occur in PCR batches, in most cases direct sequencing is not possible. For this reason, for separation and amplification, the PCR products were incorporated into bacterial vectors and the plasmids were subsequently introduced into competent cells. This was carried out by means of the TOPO TA Cloning Kit, as described below.
In order to obtain cultures that multiply only a particular plasmid, per batch 8 individual colonies were taken with sterile pipette tips from the plates obtained in chapter 2.7.2, and used for inoculating in each case 1.5 ml LB/amp medium. These were cultivated overnight at 37° C. on the shaker.
After completion of cultivation, 1 ml per culture was taken, transferred into a clean 1.5 ml tube or—depending on the number of samples—into a 96-well plate. The cells were then centrifuged off for 15 minutes at 4° C. and 3,000 rpm, and the supernatant fluid was removed. Subsequently, plasmid isolation was carried out with the QIAprep 8 Turbo Miniprep Kit according to the instructions, using the QIAvac 6S apparatus as described below.
In order to separate the inserts from the bacterial vector, and to check them, restriction digestion was carried out according to the following pipetting schema:
The restriction digestion was incubated for 1 hour at 37° C., and subsequently checked with a 1% agarose/EtBr gel.
In order to be able to examine the PCR products which had been amplified by means of bacterial plasmids for chymase sequences, their sequences were now established.
Due to the restriction digestion, it was possible to identify batches with different inserts, so that we did not sequence identical inserts several times over. For the sequencing, it was first of all necessary to carry out a chain termination synthesis with fluorescence-marked ddNTPs. These were available together with the reaction buffer and the normal dNTPS in the form of a ready mix called Big Dye (commercially attainable, Applied BioSystems (ABI), Switzerland Applied Biosystems, Rotkreuz Branch, Grundstrasse 10, CH-6343 Rotkreuz, Switzerland).
The following volumes were used for the sequencing:
The primer M13-R was used for the sequencings, since this binds immediately outside the multicloning site, and thus each insert could be sequenced completely independently of its type.
The chain termination synthesis proceeded according to the following program:
Subsequently the batches were freed of the remaining fluorescent dyes by means of the Dye Ex 2.0 kit, according to the instructions, as these would interfere with the sequencing.
The purified samples were then sequenced automatically using a sequencer, and the base sequences that were obtained were compared via a database search with all known DNA molecules. Furthermore, likewise via a database search, from the DNA sequences that were obtained, the corresponding amino acid sequences of the resulting proteins were determined.
With the aid of the first DNA part-sequence from guinea pigs, primers specific to this were to be developed. For this, those parts of the guinea pig sequence were selected that differed most clearly from the sequences of the comparison DNA of hamsters and humans, and primers of a 30 base length were developed. These primers were then produced by a contract firm.
The primers used for the subsequent PCRs are shown in
The task was once more to carry out a wide-ranging search for chymase sequences in guinea pig RNA. For this, the most varied primers that had been developed or used to date were combined with one another; amongst other things, hamster chymase primer was used again, and also the oligo (dT) primer as well as the universal adapter primer (UAP, Invitrogen). The cDNA solutions that were created with the primers oligo (dT) and adapter primer were used as templates. For the batches F and J, only the adapter primer template was used, and for the batches G and K the oligo (dT) primer template was used. All other batches were produced for both templates. In addition, batches G and K were exposed to an annealing temperature of 45° C., whereas for the others this was 58° C.
Batches were prepared with these primer combinations:
The batches were composed as follows:
The PCR proceeded according to the following program:
The PCR batches were checked with a 1% agarose/EtBr gel.
In order to have sufficient material available for investigation, selected promising PCR products were to be multiplied via transformation and bacterial cultures, and subsequently investigated for possible guinea pig sequences. For this, the batches FAP, JAP, HdI, HAP, IdI, IAP, LdI and LAP were selected.
The PCR products that were obtained and were purified by means of the QIAquick Purification Kit were once again incorporated into bacterial vectors by means of the TOPO TA cloning kit, and introduced into competent cells by means of transformation. The resulting LB/amp plates were incubated overnight at 37° C. in the incubator.
For each batch, 6 individual colonies were taken from the plates resulting from item 2.10.1, by means of sterile pipette tips. These were first of all dipped several times into a PCR batch. In the course of this, bacteria were released which in the subsequent PCR were killed off and disintegrated in the first 95° C. step, so that the plasmids contained in them could serve as templates.
The batches that were produced were composed as follows:
The PCR proceeded according to the following program:
Furthermore, the tips were then used as usual to inoculate in each case 1.5 ml LB/amp medium in a 48-well plate.
Cultivation of the liquid cultures took place overnight at 37° C. on the shaker. Subsequently, the plasmids were isolated as described above, using the QIAprep 8 Turbo Miniprep Kit.
Checking the Colony PCR
Through the colony PCR, carried out in parallel with the cultivation, the intention was to make it possible at an early stage to make a statement about the inserts contained in the liquid cultures, and thus to be able to make a pre-selection of the cultures to be processed further. The results of the colony PCR were investigated by means of a 1% agarose/EtBr gel.
Plasmids were isolated by means of the QIAprep 8 Turbo Miniprep Kit.
For the sequencing of the plasmids from the batches listed above, it was necessary to carry out chain termination synthesis with fluorescent-marked ddNTPs beforehand.
The batches are composed as follows:
The sequencing PCR proceeded according to the following program:
Subsequently, the batches were freed of the remaining fluorescent dye by means of the Dye Ex 2.0 kit, as these would interfere with the sequencing. The purified samples were then sequenced automatically by means of a sequencer, and the sequences that were obtained were compared via a database search with all known DNA molecules. Furthermore, likewise via a database search, from the DNA sequences that were obtained, the corresponding amino acid sequences of the resulting proteins were determined.
The guinea pig sequence thus obtained is shown in
The sequences of putative guinea pig chymase that had been discovered and sequenced so far corresponded, in the case of the already fully known sequence for hamster chymase, roughly to the nucleic acid sequence that codes mature chymase. The task was now to determine, starting out from this part sequence, the rest of the chymase in the 5′ direction. This was to be carried out via a so-called 5′ RACE.
New specific primers were selected on the basis of the newly determined sequence (table 7). These primers were produced by a contract firm. (Operon Biotechnologies GmbH, Nattermannallee 1, D-50829 Cologne, Germany).
The position of the primers is shown in
In order to achieve the best possible results and to be able to exclude any degeneration of the templates, 4 different cDNA templates were produced through the use of two different kits, as described below.
Transcriptor First Strand cDNA Synthesis Kit
5′ RACE Kit for Rapid Amplification of cDNA Ends
The quantities of cDNA that were obtained in Example 8.2 above were multiplied via the PCR described below.
For the PCR, the volumes listed here were pipetted together on ice:
These primer combinations were used here:
For the batch with the cDNA 2, in which the UAP was used, the 3′ RACE took place, and for the batch with cDNA 4 correspondingly the 5′ RACE.
The PCR proceeded according to the following program:
The samples were then checked with a 1% agarose/EtBr gel.
Since simple PCR was mostly insufficient for producing visible amounts in this obtaining of cDNA, a further PCR was carried out, which in this connection was referred to as “nested amplification”.
For this, the following volumes were pipetted together:
These primer combinations were used here:
The amplification products were subsequently checked with a 1% agarose/EtBr gel.
The products of the nested amplification are subsequently cloned, as described above, multiplied and sequenced.
Recombinant guinea pig chymase is expressed by means of the cloned sequence (Seq ID No. 1) in a suitable expression system, e.g. Pichia pastoris, and purified (Nakabuko et al, 2000, Yeast 16(4), 315-23; Takai et al., 1997, Clin. Chim. Acta 265, 13-20; Schechter et al., 1993, J. Biol. Chem. 268, 23626-23633), which are hereby incorporated by reference in their entirety.
The recombinant guinea pig chymase was expressed in BL21(DE3) cells as an N-terminal fusion protein with a 26-amino acid polypeptide carrying a 6×His tag and a enterokinase cleavage site. Since the protein was expressed forming insoluble inclusion bodies it had to be renatured. Isolated and purified inclusion bodies were modified under denatured conditions with glutathione and then dissolved in 6 M guanidine HCl, 20 mM EDTA pH 4.5. Refolding was done by a 1:100 dilution of the protein solution into a large volume of refolding buffer: 50 mM Tris/HCl pH 8.0, 750 mM arginine, 1 mM EDTA, 2 mM cysteine. Alkaline pH was chosen to promote thiolate anion formation and hence disulphide exchange. The preparation was kept at 4° C. for two days. Due to the high ionic strength of the refolding solution, the whole preparation had to be concentrated by ultrafiltartion and dialysed prior to a first chromatography. The dialysis buffer contained 50 mM Tris/HCl, 300 mM NaCl and 1 mM β-mercaptoethanol. Raw and still inactive chymase could be obtained by a chromatography on Ni—NTA using the same buffer and a gradient of imidazole to elute the protein from the column. Then the enzyme was activated by cleaving off the N-terminal 6×His tag with enterokinase. Thereby the mature guinea pig chymase with the correct N-terminus was formed. However, beside activation partial self digestion of the protein was also observed. The activated enzyme was twice chromatographed on Superdex 75, first in 50 mM Tris/HCl pH 8.0, 300 mM NaCl, 1 mM TCEP, 10% glycerol and then in 50 mM MES, 300 mM NaCl, 1 mM TCEP, 10% glycerol in a slightly acidic pH of 5.5 because chymase is described to be inactive at that pH (corresponds to the pH in mast cells, where active chymase is stored). Finally, as shown by SDS-PAGE >98% pure protein was obtained. The purified protein was monomeric as detected by analytical ultracentrifugation and highly active in a fluorometric assay.
The activity of guinea pig chymase is measured using familiar methods known to one of ordinary skill in the art, (Muramatsu et al., 2000, J. Biol. Chem. 275, page 5546; Uehara et al., 1999, Hypertension 35, page 57; Takai et al., 1997, Clin. Chim. Acta 265, p. 15), which are hereby incorporated by reference in their entirety.
For the chymase a substrate was chosen containing the 4 amino acid peptide AAPF as a standard substrate for chymotrypsin like compounds (succinyl-Ala-Ala-Pro-Phe-[7-amino-4-methylcoumarin]; Lockhart B E, et al., “Recombinant human mast-cell chymase: an improved procedure for expression in Pichia pastoris and purification of the highly active enzyme.” Biotechnol Appl Biochem. published as immediate publication 26 May 2004 as manuscript BA20040074)). The peptide was synthesized with a purity of 95% from Bachem, Bubendorf, Switzerland). Chymase purified from human skin mast cells was obtained from Calbiochem (Merck Biosciences, San Diego, Calif., USA). The assay buffer was 0.15 M NaCl, 0.05M, Tris HCl, 0.05% CHAPS, 0.1 mg/ml Heparin (Heparin sodium, Sigma, porcine intestinal mucosa), 0.02mM AAPF-substrate, 1 nM Chymase at pH 7.4. The assay was performed in 96-well plates (Packard Optiplate), with a 0.05 ml volume at room temperature. Chymase activity was indicated by the initial rate of increase in fluorescence at 340/440 nm (excitation/emission) from free 7-amino-4-methylcoumarin released from the substrate. Inhibition of the activity by inhibitory compounds was read after 30 min pre-incubation with the chymase at room temperature in assay buffer without AAPF-substrate. The assay was then started by addition of the indicated concentration of AAPF-substrate.
| Number | Date | Country | Kind |
|---|---|---|---|
| 05106404.6 | Jul 2005 | EP | regional |
