WO2001088530A2 - Verfahren zum selektieren von inhibitoren für enzyme - Google Patents
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- WO2001088530A2 WO2001088530A2 PCT/EP2001/005661 EP0105661W WO0188530A2 WO 2001088530 A2 WO2001088530 A2 WO 2001088530A2 EP 0105661 W EP0105661 W EP 0105661W WO 0188530 A2 WO0188530 A2 WO 0188530A2
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
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- the invention relates to a method for selecting inhibitors for enzymes and the use of the selected inhibitor as a therapeutic and / or prophylactic agent, a mutant that can be used in the method and a method for testing an inhibitor for its specificity in a model system.
- the object of the present invention is therefore to provide a method for selecting inhibitors which is simple, less time-consuming and inexpensive.
- the solution to this problem is a method for selecting inhibitors, comprising the steps a) Determination of an inhibitor-specific but not substrate-specific binding site in a wt (wild-type) enzyme, b) Replacement of at least one amino acid at the binding site of the wt enzyme determined as inhibitor-specific by an amino acid different therefrom, whereby a mutant of the wt enzyme is obtained, c) checking the mutants obtained in step b) for enzyme activity and selecting the active mutants, d) checking at least one substance with the wt enzyme and the mutant selected in step c), and e) selecting the substance as an inhibitor if it inhibits the wt enzyme, but not the mutant selected in step c).
- Another object of the invention is to provide mutants for use in the method according to the invention.
- the solution to this problem is a mutant of a wt enzyme, obtainable by performing steps a) and b) of the method according to the invention.
- Another object of the invention is to provide a simple and safe method for testing an inhibitor for biological effects which are specific for the interaction between the inhibitor and the inhibitor-specific binding site.
- FIG. 1 shows a representation of the binding mode of PP1 in comparison to adenosine at the binding site of the Src kinase Hck;
- FIG. 5 shows a schematic representation of the procedure for a selection of the mutants of the Src kinase Hck to be produced at positions 338 and 403;
- Figure 6 is a summary of the amino acid substitutions made at positions 338 and 403 of Src kinase Hck, with the mutations introduced gradually restrict access to the hydrophobic PP1 binding pocket;
- FIG. 7 shows a schematic representation of the procedure for the selection of the mutants of the protein kinase Abi to be produced at positions 315 and 380;
- FIG. 11 shows a schematic illustration of the selection of specific inhibitors with the aid of mutated forms of an enzyme
- 16 shows a schematic representation of the biological testing of an inhibitor with the aid of a "knock-in" animal model which expresses an inhibitor-resistant mutant instead of the wt enzyme;
- Figure 17 is two graphs showing the proliferation of STI571 or PP1-treated 32D cells expressing either wt Bcr-AbI or the mutant Bcr-AbI T315M in the absence of interleukin-3 as a survival factor, with the extent of proliferation as absolute cell number is shown;
- Figure 18 two graphs showing the survival of STI571 or PP1 treated 32D cells expressing either wt Bcr-AbI or the mutant Bcr-AbI T315M in the absence of interleukin-3 as a survival factor, the extent of which Cell death is shown as a percent of Annexin V positive cells;
- FIGS. 17 and 18 show a schematic illustration of the results from FIGS. 17 and 18.
- the method relates to the selection of inhibitors with optimal binding properties to a specific, selected enzyme, specific inhibitors being found.
- an inhibitor-specific but not substrate-specific binding site in a wt enzyme is determined. This determination can be made by analyzing the crystal structure of a wt enzyme for its substrate-specific binding site and the steric arrangement of a known inhibitor. tors for this wt enzyme can be performed. As a result, the binding site that is specific only for the inhibitor and not for the substrate can be found. The determination of an inhibitor-specific but not substrate-specific binding site can also be carried out without cocrystallization of the wt enzyme with a known inhibitor, that is to say only based on the crystallization of the enzyme with its substrate. In addition, instead of the crystal structure, a tertiary structure of a wt enzyme determined by calculation based on the amino acid sequence can also be used.
- the enzyme used in step a) is preferably selected from the group consisting of protein kinases, proteases and phosphatases.
- the protein kinase is preferably selected from the group consisting of the Src kinases Src, Lyn, Fyn, Hck, Lck, Blk, Yes, Yrk, Fgr, the kinases ZAP70, BTK, Tee, Jak1, Jak2, PKA, VEGF family -, PDGF family and EGF family receptor kinases, MAP kinases, c-Abl and cyclin-dependent kinases, in particular the Src kinases Hck or Lyn and the kinase c-Abl.
- the Src kinase Hck and the kinase Abi are particularly preferably used in step a).
- the known inhibitor PP1 with the Src kinase Hck and the known inhibitors PP1 and STI571 with the tyrosine kinase Abi are used, which shows that the method according to the invention can be used to find specific inhibitors for the enzymes
- the crystal structures of Src kinase Hck published by Schindler et al., Molecular Cell, 1999, 3, pp. 638-648 and by Zhu et al., Structure, 1999, 7, pp. 651-616 were complexed used with PP1 and the Src kinase Lck in complex with PP2 to determine the structural requirements for the specificity of the inhibitors for the Src kinases.
- the binding mode of the pyrazolopyrimidine scaffold roughly corresponds to that of the purine scaffold of ATP.
- the 4-methylphenyl residue of PP1 or the 4-chlorophenyl residue of PP2 into a hydrophobic pocket, the entry of which is limited by the amino acid side chains of lysine (K) at position 295, valine (V) at position 323, threonine (T) at position 338 and alanine (A) at position 403 , as shown in Figure 1.
- This hydrophobic pocket is therefore an inhibitor-specific but not substrate-specific binding site.
- the hydrophobic pocket itself is limited, inter alia, by the amino acids methionine at position 314, leucine at position 325, isoleucine at position 336, aspartate at position 404 and phenylalanine at position 405, as also shown in FIG. 1.
- amino acids T338 and A403 differences in the amino acid sequence affect the space and hydrophobicity ratios within the hydrophobic pocket and thus the binding of the inhibitors to the binding site.
- a potential inhibitor-specific binding site in the tyrosine kinase Abi was identified based on sequence homology comparisons and a calculated model. This is a pocket that is 100% homologous to the above-described hydrophobic pocket of Src Kinase Hck, as can be seen in FIGS. 4 and 5.
- the entrance area to this day see is formed by the amino acids Lysin271, Val299, Thr315 and Ala380.
- Amino acid alignments with other tyrosine kinases showed that, as already described for Hck, they have different amino acids, in particular at the positions homologous to T315 and A380, as shown in FIG. 4. These amino acids have a longer side chain, so that access to the pocket described above is critically restricted or blocked.
- step b) of the method according to the invention at least one amino acid at the binding site determined as inhibitor-specific is replaced by an amino acid different therefrom.
- the number of mutated positions and the type of amino acids introduced in the exchange depend on the structure of the binding site determined as inhibitor-specific. As a rule, those amino acid positions that have the greatest possible variability within an enzyme family are changed, i.e. are occupied as differently as possible with amino acids within an enzyme family.
- step b) the amino acid is preferably replaced by a more sterically complex, more hydrophobic, more hydrophilic, more basic or more acidic amino acid.
- amino acids for exchange which are sterically more complex than the exchanged amino acid.
- more sterically complex means that the new amino acid has a longer or more voluminous side chain than the replaced one. It is preferable to carry out several amino acid exchanges per position, so that a gradual Change in the spatial conditions in the area of an inhibitor-specific binding site arises. Such mutants are grouped into libraries
- the hydrophobicity or hydrophilicity of this binding site is changed. If, for example, the binding site is built up with predominantly hydrophobic amino acids, the hydrophobicity can be changed in that the new amino acid is hydrophilic.
- a wide variety of amino acid substitutions were carried out in positions 338 and 403 of the Src kinase Hck, as shown in FIG. 5.
- the numbering of the positions refers to the homologous position in c-Src from chicken.
- the new amino acids are sterically more complex than those present in the wt Src kinase Hck. As determined in the analysis of the crystal structure and shown in FIG. 1, these positions are at the entrance of the hydrophobic pocket, which is the inhibitor-specific binding site. Because the new amino acids are sterically more complex, access to the hydrophobic pocket is reduced.
- the selection of the amino acids introduced by exchange is based on sequence homology comparisons, i.e. only those amino acids were introduced which occur in the homologous position in other kinases.
- Kinases from the same species, for example humans, are preferably taken into account.
- the Src kinase Hck was used in step b), preferably replacing threonine at position 338 and alanine at position 403 by a different amino acid.
- threonine at position 338 is replaced by an amino acid selected from the group consisting of valine, leucine, isoleucine, methionine, glutamine and phenylalanine, and alanine at position 403 by an amino acid selected from the group consisting of Serine, cysteine and threonine, these are the amino acids that are at the homo position in other kinases be found.
- the corresponding mutations lead to a gradual narrowing of access to the hydrophobic pocket, as shown in FIG. 6.
- leucine at position 325 and isoleucine at position 336 can be replaced by phenylalanine and / or threonine.
- the hydrophobicity of the hydrophobic pocket is reduced on the one hand by substitution with threonine and the space inside the pocket is reduced by substitution with phenylalanine.
- a mutation of aspartate at position 404 and phenylalanine at position 405 does not make sense because of their important catalytic function.
- Abi threonine at position 315 was replaced by the amino acids valine, leucine, isoleucine, methionine, glutamine and phenylalanine.
- Abi alanine at position 380 was exchanged for the amino acids serine, cysteine and threonine. These are the amino acids found at the homo position in other kinases as shown in Figure 7. The corresponding mutations result in a gradual narrowing of access to the potential inhibitor binding site pocket, as shown in FIG. 8.
- the exchange of the amino acids of the enzyme can be carried out using conventional mutagenesis methods, such as PCR mutagenesis.
- PCR mutagenesis the gene to be mutated is inserted into a cloning vector.
- DNA primers that are muta- containing gene codons the corresponding mutation is introduced into the gene using a standard PCR reaction.
- the mutated gene is then amplified in bacteria and the mutation confirmed by sequencing.
- mutants of the wt enzyme are obtained which are sterically modified at the inhibitor-specific binding sites or in relation to their hydrophilicity, hydrophobicity, basicity or acidity compared to the wt enzyme.
- step c) of the method the mutants obtained in step b) are checked to determine whether they still show the enzyme activity of the wt enzyme. Then the mutants are selected in which the exchange of the amino acids in step b) does not influence the enzyme activity, i.e. the active mutants.
- mutants of the Src kinase Hck was tested, as shown in FIG. 9.
- threonine at position 338 was replaced by valine, leucine, isoleucine, methionine, glutamine and phenylalanine, and alanine at position 403 by serine, leucine and threonine.
- the respective mutants were expressed in Cos7 cells.
- T338V, T338Q, T338F, A403S and A403C show approximately the same activity as wt Hck.
- A403T showed slightly less activity than wt Hck. It can thus be seen from FIG. 9 that an exchange of threonine at position 338 by leucine, isoleucine and methionine leads to hyperactivation and an exchange by valine, glutamine and phenylalanine leads to an activity comparable to the wt Src kinase Hck. The exchange of alanine at position 403 to serine and cysteine also leads to an activity comparable to wt Src kinase Hck. An exchange for threonine leads to a slight decrease in kinase activity
- mutants of the Abi kinase or the Abi leukemia-inducing subfrom, Bcr-AbI was tested, as shown in FIG.
- threonine at position 315 was exchanged for valine, leucine, isoleucine, methionine, glutamine and phenylalanine.
- the respective mutants were expressed in Cos7 cells.
- step c) all mutants which have enzymatic activations can be selected for carrying out steps d) and e) of the method according to the invention. did own. It is preferred to select as many mutants as possible with a gradual gradation of the changes at the binding site specific for the inhibitor. If a mutant shows no enzyme activity anymore, it can be assumed that by changing an amino acid at the inhibitor-specific binding site the tertiary structure of the enzyme was influenced so strongly that this mutant is no longer suitable in the subsequent steps to be used to select a specific inhibitor for the wt enzyme. The mutant selected in step c) thus has the greatest possible structural similarity with the wt enzyme, with the difference that the most gradual possible change was made at the binding site of the enzyme specific for the inhibitor.
- mutants T338V, T338L, T338I, T338M, T338Q, T338F, A403S, A403C and A403T were selected for the Src kinase Hck. These mutants all have sufficient kinase activity to be used for steps d) and c).
- mutants T315V, T315L, T315I, T315M, T315Q and T315F were selected for the kinase Abi. These mutants all have sufficient kinase activity to be used for steps d) and c).
- the mutants selected in step c) of the method according to the invention can additionally be checked to determine whether they are inhibited by the known inhibitor. The mutants that are no longer inhibited by the known inhibitor are then selected.
- step d) of the method according to the invention at least one substance is tested with the wt enzyme and the mutants selected in step c).
- a substance library consisting of a wide variety of substances is preferably tested simultaneously. This distinguishes 3 different classes of substances, namely (1) substances that neither inhibit the wt enzyme nor the mutant selected in step c) (no inhibitor), (2) substances that both the wt enzyme and those in step c) inhibit selected mutant (unspecific inhibitor) and (3) substances which only inhibit the wt enzyme, but not the mutant selected in step c), as shown schematically in FIG. 11. Those substances (3) which only inhibit the wt enzyme but not the mutant selected in step c) are then selected as inhibitors.
- a substance or substance library is preferably tested with a large number of mutants selected in step c), preferably with at least 2, in particular with at least 5 and particularly preferably with at least 10 mutants.
- the substance that inhibits only the wt enzyme and none of the selected mutants is preferably selected.
- the known Src inhibitor PP1 can be selected using the method according to the invention.
- the tyrosine phosphorylation activity of the mutants which was replaced by the replacement of threonine at position 338 by valine, leucine, isoleucine, methionine, glutamine and phenylalanine and by the exchange of alanine at position 403 by serine, cysteine and threonine in the wt Src- Kinase Hck were tested with and without the addition of inhibitor PP1, as shown in FIGS. 12 and 13. It can be seen from FIGS.
- mutants threonine to leucine, isoleucine, methionine, glutamine and phenylalanine were not inhibited by PP1.
- the mutants threonine 315 to valine and alanine 403 to serine, cysteine and threonine were inhibited by PP1 like the wt form of Hck.
- PP1 is selected as a substance which binds into the hydrophobic pocket, ie the inhibitor-specific binding site.
- the mutants were expressed in Cos7 cells, with the difference that they were additionally incubated with PP1 before the lysis.
- wt Hck leads to a significant increase in the tyrosine phosphorylation of numerous cellular proteins. Incubation of the cells with 100 ⁇ M PP1 prior to lysis abolishes or significantly reduces phosphorylation induced by wt Hck. The same applies to the mutants T338V, A403S, A403C and A 403T. In contrast, the phosphorylation induced by the mutants T338L, T338I, T338M, T338Q and T338F remain largely unchanged even after incubation with PP1. These mutants are therefore resistant to PP1 inhibition.
- Resistance to PP1 is therefore induced by sufficiently restricting access to the hydrophobic PP1 binding pocket by extending the amino acid side chain at position 338, without losing the basic activity of the enzyme. This result confirms the importance of the hydrophobic PP1 binding pocket for the binding of PP1. Conversely, restricting access to the hydrophobic "inhibitor binding pocket" by extending the amino acid side chain at position 403 does not lead to resistance to PP1.
- Table 1 summarizes examples of amino acid exchanges carried out in the kinase Hck and the results of the tests carried out in steps c) and d).
- the tyrosine phosphorylation activity of the tyrosine kinase Abi or its leukemia-inducing form, Bcr-AbI, and the mutants of Bcr-AbI at position 315 became valine, leucine, isoleucine, methionine, glutamine and phenylalanine with and without addition of known kinase -Inhibitors tested.
- PP1 and STI571 were used as inhibitors. This was intended to determine inhibitors which bind into the potential “inhibitor binding pocket” of the Abl kinase and thus have the greatest possible specificity with respect to other protein kinases.
- the mutants were expressed in Cos7 cells, with the difference that they were additionally incubated with PPI or STI571 before lysis.
- FIG. 14 representatively shows the results for the incubation of Bcr-AbI wt and the mutants of Bcr-AbI at position 315 to give valine, leucine, isoleucine, methionine, glutamine and phenylalanine with PP1.
- the expression of Bcr-AbI wt leads to a significant increase in the tyrosine phosphorylation of numerous cellular proteins. Incubation of the cells before lysis with the inhibitor PP1 abolishes or significantly reduces the phosphorylation induced by Bcr-AbI wt. The tyrosine phosphorylation induced by the mutant T315V is also abolished.
- Table 2 shows the results of the inhibition of Bcr-AbI and mutants of Bcr-AbI at positions 315 and 380 with PP1 and STI571.
- PP1 also inhibits the mutants T315V, A380S, A380C and A380T.
- the mutants T315L, T315I, T315M, T315Q and T315F could no longer be inhibited by PP1.
- STI571 only inhibits the mutants A380S and A380C.
- the mutants T315V, T315L, T315I, T315M, T315Q and T315F and A380T could no longer be inhibited.
- step e) of the method the substance is selected as an inhibitor which inhibits the wt enzyme, but not the mutants selected in step c), as shown in FIG. 11.
- Those substances which inhibit the wt enzyme are preferably selected, but not the mutant with the smallest structural change, that is to say, for example, with only a slight extension of the amino acid side chain at a position identified as a specificity determinant.
- PP1 is selected as an inhibitor for the Src kinase Hck and STI571 as an inhibitor for Abi.
- STI571 is selected as PP1 inhibitor because it, unlike PP1, can no longer inhibit the mutants T315V and A380T. Based on the gradual changes that were made by the respective mutations at positions 315 and 380 in Abi (see FIG. 8), STI571 fills the entrance to the hydrophobic binding pocket better.
- the method described above simplifies the targeted screening for lead structures which are suitable as specific inhibitors for enzymes, such as Src kinases or Abi-kinase.
- enzymes such as Src kinases or Abi-kinase.
- the screening process is considerably simplified, since the inhibitor-resistant mutant is now screened and not against a variety of potentially cross-inhibitable other kinases.
- the development times and costs for such an experimental set-up are significantly lower overall.
- a selection for structural features which give potential inhibitors the highest possible specificity with respect to other potential target molecules can already take place at the lead identification stage.
- the known inhibitors for these two kinases are successfully recognized using the method according to the invention.
- a comparison of the method according to the invention when screening PP1 and STI571 against mutants and the wt enzyme Abi shows that the method according to the invention is used to select the more specific of the two inhibitors, STI571.
- inhibitors are found which have a high specificity compared to the inhibition of certain enzymes. These can be used in a variety of areas. In particular, these can be used as therapeutic and / or prophylactic agents in the treatment of diseases such as cancer, allergies, rejection reactions in transplantations and / or osteoporosis. Cancer diseases, such as leukemia or solid tumors, are preferably treated with the agent according to the invention.
- a method for testing an inhibitor selected with the method according to the invention for biological effects which are specific for the interaction between the inhibitor and the inhibitor-specific binding site comprising the steps
- the model system used in the method according to the invention is preferably selected from the group consisting of cell lines, microorganisms and animals. Mice, rats or rabbits can be used as animals. Cell lines 32D and BaF3, both model systems for leukemia, are preferably used as cell lines.
- the effects determined in step 2) are preferably selected from the group consisting of therapeutic effects, acute and subacute organ toxicity, non-therapeutic immunosuppression and lethal effects.
- inhibitor-resistant mutants can thus be used for further, biological validation of selected and optimized inhibitors or inhibitor lead structures.
- the method according to the invention can be used to differentiate enzyme-specific effects of the inhibitor, and thus desired effects, such as, for example, therapeutic effects, from enzyme-independent effects of the inhibitor, that is to say undesired effects.
- these mutants can be used to differentiate between enzyme-specific and enzyme-independent effects of the inhibitor, in particular between enzyme-specific and enzyme-independent side effects.
- a model system for example a mouse
- the model system can also be a cell line.
- an inhibitor-resistant allele are expressed in a "knock-in" mouse.
- the corresponding gene product (IR) can no longer be inhibited by the inhibitor and can therefore also perform its function in its presence.
- Embryonic stem cells are cultivated to produce a “knock-in” mouse which can be used according to the invention.
- a transfer vector is introduced into these cells which, in addition to the gene of interest, also its regions flanking the genome and a selection marker , ie a resistance gene, through homologous recombination there is an exchange of the wt gene for the “knock-in” gene.
- those ES cells are then selected in which the desired gene exchange has taken place.
- the ES cells selected in this way are injected into mouse blastocysts and these are implanted in sham pregnant mice. In this way, chimeras are obtained, which are finally used by crossbreeding to produce genetically pure progeny.
- I in the decision-making aid which the method according to the invention provides as part of the development of an inhibitor.
- a large number of enzyme-independent, undesirable side effects occur, this speaks for an additional interaction of the inhibitor with a further target structure within the organism, which is different from the therapeutic target structure. Accordingly, either the specificity of the inhibitor must be increased by appropriate modifications, or another substance must be found which does not lead to undesired, enzyme-independent side effects.
- Kicks If a large number of enzyme-dependent side effects occur, the enzyme is challenged as a target structure that can be attacked by therapy. Accordingly, a better, therapeutically usable enzyme must be sought for the corresponding disease. This decision-making and development process can save high costs for later, unsuccessful clinical studies.
- the enzyme or target structure specificity of the therapeutic response is clarified with the method according to the invention.
- Knowledge of the enzyme or target structure specificity of the therapeutic response can in turn significantly accelerate the approval process for a drug containing the inhibitor.
- cell culture models can also be used for the biological validation of inhibitors with the aid of the method according to the invention.
- Bcr-AbI is a constitutively active tyrosine kinase that induces various forms of leukemia in animal models and in humans.
- 32D cells were transfected with a plasmid by means of electroporation, that is to say with the aid of a current pulse.
- This plasmid encoded either wt Bcr-AbI or e.g. for the mutant T315M.
- these plasmids carry a resistance marker, in this case a puromycin resistance gene. Transfected cells were therefore selected with the help of puromycin. After selection, some of the cells were lysed and the expression of Bcr-AbI was detected by means of western blot analysis.
- interleukin-3 was withdrawn from the cells as an essential survival and growth factor. While untransfected 32D cells stop proliferating with interleukin-3 withdrawal and die within 24 hours, cells with wt Bcr-AbI as well Cells that had been transfected with a mutant of Bcr-AbI at position 315, for example T315M, survive and proliferate even in the absence of interleukin-3 in the culture medium (FIGS. 17 and 18), to the same extent. This shows that the mutation of Bcr-AbI at position 315, for example to methionine, does not cancel or reduce the leukemogenic potency of the kinase (FIG. 17).
- STI571 cannot inhibit Bcr-AbI because the extension of the amino acid side chain at position 315 blocks access to the inhibitor-specific binding site.
- the absence of a biological effect of STI571 in these cells therefore proves that Bcr-AbI is the only relevant target molecule of STI571 under these conditions.
- the effect of STI571 is therefore "target molecule" -specific. This suggests a low toxicity of the substance.
- STI571 is selected here as Abispecific inhibitor, because its biological effect on 32D cells expressing Bcr-Abl is purely “target molecule-specific”. Although PP1 also inhibits Bcr-AbI and leads to cell death, this effect is not entirely “target molecule” -specific, so that this inhibitor is expected to be more toxic in vivo. In fact, STI571 is currently being used in clinical trials Studies tested without any signs of relevant toxicity.
- the method according to the invention can therefore also be used for the detailed molecular analysis of enzyme-unspecific effects.
- a method for carrying out the method according to the invention is described in more detail below.
- Cos7 cells were cultured in Dulbecco's modified Eagle's Medium (DMEM) enriched with 10% fetal calf serum (FKS).
- DMEM Dulbecco's modified Eagle's Medium
- FKS fetal calf serum
- the cDNA of the human Hck gene was cloned into the vector pUC18.
- This plasmid then served as a template for a mutagenic PCR reaction.
- the PCR was performed using mutagenic primers that were phosphorylated at their 5 'ends and designed to bind to immediately adjacent regions. With the aid of standard settings (annealing temperature corresponding to the primer), the complete plasmid was amplified, Pfu (Promega) being used as the polymerase.
- the linear PCR amplificate was then purified, religated to a plasmid using T4 DNA ligase and clonally propagated in bacteria. The mutants produced in this way were finally examined for the correctness of the mutation.
- the mutated Hck alleles were cloned into the EcoRI site of the vector pApuro.
- This vector is derived from pBabepuro, but has a chicken actin promoter.
- PP1 was purchased from Alexis and stored as a 25mM stock solution at 4 ° C for a maximum of 4 weeks.
- the medium was removed for lysis and the Cos7 cells were dissolved from the bottom of the culture bottle with 3 ml of trypsin-EDTA solution. The cells were then transferred to a 50 ml centrifuge tube with DMEM / 10% FCS and centrifuged.
- the cell pellet was taken up in 250 ⁇ l lysis buffer (1% NP-40, 20 mM Tris (pH 8.0), 50 mM NaCl, and 10 mM EDTA, 1 mM PMSF, 10 ⁇ g / ml aprotinin, 10 ⁇ g / ml leupeptin and 2 mM sodium orthovanadate.) and transferred to a 1.5 ml Eppendorf reaction vessel. The mixture was briefly mixed with a vortex and incubated for 30 min at 4 ° C. on an overhead rotor. The cell lysate was then centrifuged for 15 minutes at 4 ° C. and 14,000 revolutions per minute (rpm). yaws. Finally, the supernatant, which contained the cytoplasmic proteins, was transferred to a new reaction vessel.
- lysis buffer 1% NP-40, 20 mM Tris (pH 8.0), 50 mM NaCl, and 10 mM EDTA, 1 mM PMSF, 10
- the proteins of the cell lysates obtained above 80 ⁇ g of the lysate were mixed 1: 1 with 2x sample buffer and denatured for 5 minutes at 100 ° C. using SDS-polyacrylamide electrophoresis. The samples were completely loaded into the pockets of the gel. After the proteins had been separated in an electric field, they were transferred to a nitrocellulose membrane. This was then incubated for at least 1 h in a plastic dish with 15 ml of TBS, which contained 5% skimmed milk powder. The membrane was then rinsed 2-3 times with TBS.
- the membrane was then incubated for 2 to 18 h with the primary antibody (anti-phosphotyrosine PY99 from Santa Cruz Biotch., Anti-Hck N-30 from Santa Cruz Biotech, in dilutions of 1: 1000 in each case).
- the antibodies were diluted in 15 ml TBS / 1% skim milk powder.
- the membrane was then three times 5 min. washed with TBS.
- the membrane was incubated for 1 h with secondary antibody (donkey-anti-mouse or donkey-anti-rabbit antibody coupled with Merrettich-Peroxidas in a dilution of 1: 2000; both antibodies from Amer- sham) and then again three times for 5 min rinsed with TBS.
- ECLTM detection reagents 1 and 2 were mixed and poured onto the membrane in a plastic dish in the dark room. After exactly 1 min. the detection solution was poured off, the membrane removed, drained well and covered with Saran packaging film so that there were no bubbles. Finally, ECLTM hyperfilms were put on for 3 to 60 seconds and developed in a suitable developing apparatus (Agfa).
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
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IL15290601A IL152906A0 (en) | 2000-05-17 | 2001-05-17 | Method for selecting enzyme inhibitors |
CA002409809A CA2409809A1 (en) | 2000-05-17 | 2001-05-17 | Method for selecting enzyme inhibitors |
AT01933986T ATE288081T1 (de) | 2000-05-17 | 2001-05-17 | Verfahren zum selektieren von inhibitoren für enzyme |
AU2001260314A AU2001260314A1 (en) | 2000-05-17 | 2001-05-17 | Method for selecting enzyme inhibitors |
EP01933986A EP1282827B1 (de) | 2000-05-17 | 2001-05-17 | Verfahren zum selektieren von inhibitoren für enzyme |
DE50105206T DE50105206D1 (de) | 2000-05-17 | 2001-05-17 | Verfahren zum selektieren von inhibitoren für enzyme |
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DE10024174A DE10024174A1 (de) | 2000-05-17 | 2000-05-17 | Verfahren zum Selektieren von Inhibitoren für Enzyme |
DE10024174.3 | 2000-05-17 |
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WO2001088530A2 true WO2001088530A2 (de) | 2001-11-22 |
WO2001088530A3 WO2001088530A3 (de) | 2002-11-14 |
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EP (1) | EP1282827B1 (de) |
AT (1) | ATE288081T1 (de) |
AU (1) | AU2001260314A1 (de) |
CA (1) | CA2409809A1 (de) |
DE (2) | DE10024174A1 (de) |
IL (1) | IL152906A0 (de) |
WO (1) | WO2001088530A2 (de) |
Cited By (2)
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WO2004107114A2 (en) * | 2003-05-22 | 2004-12-09 | Sgx Pharmaceuticals, Inc. | Crytals and structures of c-abl tryosine knase domain |
US8697348B2 (en) | 2001-06-14 | 2014-04-15 | The Regent Of The University Of California | Mutations in the Bcr-Abl tyrosine kinase associated with resistance to STI-571 |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
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EP1541694A1 (de) * | 2003-12-12 | 2005-06-15 | Sirenade Pharmaceuticals AG | Verfahren zur Identifizierung, Auswahl und Charakterisierung von Substanzen welche die Aktivität von Kinasen der Src Familien modulieren |
DK2447252T3 (da) * | 2004-05-23 | 2020-03-16 | Hmi Medical Innovations Llc | Theramuteinmodulatorer |
US8431110B2 (en) * | 2005-05-23 | 2013-04-30 | Hmi Medical Innovations, Llc. | Compounds and method of identifying, synthesizing, optimizing and profiling protein modulators |
WO2007056177A2 (en) * | 2005-11-04 | 2007-05-18 | Bristol-Myers Squibb Pharma Company | T315a and f317i mutations of bcr-abl kinase domain |
CN103789389B (zh) | 2005-11-23 | 2017-01-04 | 杰勒德·M·豪斯 | 鉴定、合成、优化和表征蛋白调节剂的化合物和方法 |
WO2007062213A2 (en) | 2005-11-23 | 2007-05-31 | Housey Pharmaceuticals Inc | Compounds and methods of identifying, synthesizing, optimizing and profiling protein modulators |
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WO1998035048A2 (en) * | 1997-02-07 | 1998-08-13 | Princeton University | Engineered protein kinases which can utilize modified nucleotide triphosphate substrates |
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2001
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WO1998035048A2 (en) * | 1997-02-07 | 1998-08-13 | Princeton University | Engineered protein kinases which can utilize modified nucleotide triphosphate substrates |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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US8697348B2 (en) | 2001-06-14 | 2014-04-15 | The Regent Of The University Of California | Mutations in the Bcr-Abl tyrosine kinase associated with resistance to STI-571 |
US9056924B2 (en) * | 2001-06-14 | 2015-06-16 | The Regents Of The University Of California | Mutations in the BCR-ABL tyrosine kinase associated with resistance to STI-571 |
US9085644B2 (en) | 2001-06-14 | 2015-07-21 | The Regents Of The University Of California | Mutations in the Bcr-Abl tyrosine kinase associated with resistance to STI-571 |
US9994910B2 (en) | 2001-06-14 | 2018-06-12 | The Regents Of The University Of California | Mutations in the Bcr-Abl tyrosine kinase associated with resistance to STI-571 |
WO2004107114A2 (en) * | 2003-05-22 | 2004-12-09 | Sgx Pharmaceuticals, Inc. | Crytals and structures of c-abl tryosine knase domain |
WO2004107114A3 (en) * | 2003-05-22 | 2007-03-22 | Sgx Pharmaceuticals Inc | Crytals and structures of c-abl tryosine knase domain |
JP2007522792A (ja) * | 2003-05-22 | 2007-08-16 | エスジーエックス ファーマシューティカルズ,インコーポレイティド | c−Ab1チロシン・キナーゼ・ドメインの結晶及び構造 |
Also Published As
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EP1282827B1 (de) | 2005-01-26 |
WO2001088530A3 (de) | 2002-11-14 |
DE50105206D1 (de) | 2005-03-03 |
IL152906A0 (en) | 2003-06-24 |
AU2001260314A1 (en) | 2001-11-26 |
US20030162222A1 (en) | 2003-08-28 |
EP1282827A2 (de) | 2003-02-12 |
CA2409809A1 (en) | 2002-11-15 |
ATE288081T1 (de) | 2005-02-15 |
DE10024174A1 (de) | 2001-11-29 |
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