Patent application title: DETECTION OF GLEEVEC RESISTANCE
Inventors:
Brian J. Druker (Portland, OR, US)
Amie Corbin (Portland, OR, US)
Amie Corbin (Portland, OR, US)
Assignees:
Oregon Health & Science University
IPC8 Class: AC12Q168FI
USPC Class:
435 611
Class name: Measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving nucleic acid nucleic acid based assay involving a hybridization step with a nucleic acid probe, involving a single nucleotide polymorphism (snp), involving pharmacogenetics, involving genotyping, involving haplotyping, or involving detection of dna methylation gene expression
Publication date: 2013-10-17
Patent application number: 20130273542
Abstract:
The present invention relates to isolated polypeptides which comprise an
amino acid sequence consisting of a mutated functional Abl kinase domain,
said mutated functional kinase domain being resistant to inhibition of
its tyrosine kinase activity by
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)-benzamide or a salt thereof, to the use of such
polypeptides to screen for compounds which inhibit the tyrosine kinase
activity of such polypeptides, to nucleic acid molecules encoding such
polypeptides, to recombinant vectors and host cells comprising such
nucleic acid molecules and to the use of such nucleic acid molecules in
the production of such polypeptides for use in screening for compounds
which inhibit the tyrosine kinase activity of such polypeptides.Claims:
1. A method of detecting STI571 resistance in a subject, comprising:
selecting a subject undergoing STI571 therapy; obtaining a sample from
the subject; detecting a mutation in the amino acid sequence of an Abl
kinase domain of a Bcr-Abl polypeptide in the sample obtained from the
subject compared to position 255 of the Abl kinase domain of the amino
acid sequence set forth as SEQ ID NO: 2, wherein the mutation confers
resistance to kinase inhibition by STI571, thereby detecting STI571
resistance in the subject.
2. The method of claim 1, wherein the method comprises detection of an A to T point mutation that results in the substitution of valine for glutamic acid at position 255 or a G to A point mutation that results in the substitution of lysine for glutamic acid at position 255.
3. The method of claim 1, wherein detecting a mutation in the Abl kinase domain of the Bcr-Abl polypeptide comprises: determining the sequence of a nucleic acid encoding the Abl kinase domain of the Bcr-Abl polypeptide in the sample obtained from the subject; and comparing the amino acid sequence encoded by the nucleic acid with the amino acid sequence set forth as SEQ ID NO: 2; wherein a difference in the amino acid sequence of the Abl kinase domain of the Bcr-Abl polypeptide from the sample obtained from the subject compared to position 255 of the Abl kinase domain of the amino acid sequence set forth as SEQ ID NO: 2 detects an STI571 resistant Abl kinase domain polypeptide.
4. The method of claim 1, wherein determining the nucleic acid sequence comprises reverse transcriptase polymerase chain reaction.
5. The method of claim 1, wherein determining the nucleic acid sequence comprises DNA sequencing.
6. The method of claim 1 wherein the subject has a tumor.
7. The method of claim 6, wherein the tumor is a leukemia.
8. The method of claim 1, wherein detecting a mutation in an Abl kinase domain of the Bcr-Abl polypeptide comprises: determining the sequence of an Abl kinase domain in a sample from the subject; and comparing the amino acid sequence of the Abl kinase domain with position 255 of the Abl kinase domain of the amino acid sequence set forth as SEQ ID NO: 2, wherein a difference in the amino acid sequence of the Abl kinase domain from the sample from position 255 of the Abl kinase domain of the amino acid sequence set forth as SEQ ID NO: 2 detects the STI571 resistant Abl kinase domain polypeptide.
9. The method of claim 8, wherein the substitution is a substitution of valine for glutamic acid or lysine for glutamic acid at position 255.
10. The method of claim 8, wherein the subject has a tumor.
11. The method of claim 10, wherein the tumor is a leukemia.
12. A method of detecting a STI571 resistant mutation in a subject, comprising: selecting a subject for possible treatment with STI571; obtaining a sample from the subject; detecting a mutation in an amino acid sequence of the Abl kinase domain of a Bcr-Abl polypeptide in the sample obtained from the subject compared to position 255 of the Abl kinase domain of the amino acid sequence set forth as SEQ ID NO: 2, wherein the mutation confers resistance to kinase inhibition by STI571, thereby detecting an STI571 resistant mutation in the subject.
13. The method of claim 12, wherein the substitution is a substitution of valine for glutamic acid or lysine for glutamic acid at position 255.
14. The method of claim 12, wherein detecting a mutation in an Abl kinase domain of the Bcr-Abl polypeptide, comprises: determining the sequence of the Abl kinase domain in the sample from the subject; and comparing the amino acid sequence of the Abl kinase domain in the sample with position 255 of Abl kinase domain of the amino acid sequence set forth as SEQ ID NO: 2, wherein a difference in the amino acid sequence of the Abl kinase domain from the sample from position 255 of the Abl kinase domain of the amino acid sequence set forth as SEQ ID NO: 2 indicates the presence of mutant Abl kinase domain polypeptide in the subject.
15. The method of claim 12, wherein the subject has a tumor.
16. The method of claim 15, wherein the tumor is a leukemia.
17. The method of claim 16, wherein the leukemia is myelogenous leukemia.
18. The method of claim 14, wherein the subject is undergoing STI571 therapy and the method is used to detect the presence of the STI571 resistant Abl kinase domain mutation prior to clinical relapse.
19. The method of claim 7, wherein the leukemia is myelogenous leukemia.
20. The method of claim 11, wherein the leukemia is myelogenous leukemia.
21. The method of claim 1, wherein detecting the mutation in the amino acid sequence of the Abl kinase domain of the Bcr-Abl polypeptide in the sample compared to position 255 of the Abl kinase domain of the amino acid sequence set forth as SEQ ID NO: 2 detects STI571 resistance in the subject prior to clinical relapse of the subject during STI571 therapy.
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent application Ser. No. 12/803,962, filed, Jul. 9, 2010, which is a continuation of U.S. patent application Ser. No. 12/337,322, filed Dec. 17, 2008, now abandoned, which is a continuation of U.S. patent application Ser. No. 11/343,891, filed Jan. 31, 2006, now abandoned, which is a continuation of U.S. patent application Ser. No. 10/263,480, filed Oct. 3, 2002, now abandoned. U.S. patent application Ser. No. 10/263,480 claims the benefit of U.S. Provisional Application No. 60/327,387, filed Oct. 5, 2001.
FIELD OF THE INVENTION
[0002] This invention relates to isolated polypeptides which comprise an amino acid sequence consisting of a mutated functional Abl kinase domain, said mutated functional kinase domain being resistant to inhibition of its tyrosine kinase activity by N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi- perazin-1-ylmethyl)-benzamide or a salt thereof, to the use of such polypeptides to screen for compounds which inhibit the tyrosine kinase activity of such polypeptides, to nucleic add molecules encoding such polypeptides, to recombinant vectors and host cells comprising such nucleic acid molecules and to the use of such nucleic acid molecules in the production of such polypeptides for use in screening for compounds which inhibit the tyrosine kinase activity of such polypeptides.
BACKGROUND OF THE INVENTION
[0003] Bcr-Abl, a constitutively activated tyrosine kinase resulting from the formation of the Philadelphia chromosome [Nowell P. C. and Hungerford D. A., Science 132, 1497 (1960)] by reciprocal translocation between the long arms of chromosomes 9 and 22 [Rowley J. D., Nature 243, 290-293 (1973)], has been established as the characteristic molecular abnormality present in virtually all cases of chronic myeloid leukemia (CML) and up to 20 percent of adult acute lymphoblastic leukemia (ALL) [Faderl S. et al., N Engl J Med 341, 164-172 (1999); Sawyers C. L., N Engl J Med 340, 1330-1340 (1999)]. Bcr-Abl is sufficient to cause CML in mice [Daley G. Q. et al., Science 247, 824-830 (1990)] and its transforming capacity is absolutely dependent on tyrosine kinase activity [Lugo T. G. et al., Science 247, 1079 (1990)]. The compound N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi- perazin-1-ylmethyl)-benzamide (hereinafter also referred to as "STI571"; STI571 is described in EP-0 564 409 and, in the form of the methane sulfonate salt, in WO 99/03854), a competitive inhibitor at the ATP-binding site of Bcr-Abl, as well as of the receptor for platelet-derived growth factor, and c-kit tyrosine kinase [Lugo T. G. et al., Science 247, 1079 (1990)], has been shown to be capable of very rapidly reversing the clinical and hematological abnormalities of CML in chronic phase and in blast crisis as well as of Ph-chromosome-positive (Ph+) acute lymphoblastic leukemia (Ph+ ALL) [Druker B. J. et al., N Engl J Med 344, 1031-1037 (2001); Druker B. J. et al., N Engl J Med 344, 1038-1042 (2001)]. Whereas almost all chronic phase CML patients durably respond, remissions in CML blast crisis and Ph+ALL are transient, and most patients relapse after several months, despite continued therapy with STI571 [Druker B. J. et al., N Engl J Med 344, 1038-1042 (2001)]. The mechanism of resistance to STI571 is subject of intense research.
[0004] It was now surprisingly found that mutations present in the kinase domain of the Bcr-Abl gene of patients suffering from CML or Ph+ALL account for the biological resistance of these patients towards STI571 treatment in that said mutations lead to resistance of the Bcr-Abl tyrosine kinase towards inhibition by STI571.
[0005] These findings are extremely valuable in e.g. finding new compounds or combinations of compounds which are capable to overcome resistance towards treatment with STI571. Moreover, knowledge of such mutations is also very useful in the diagnosis of Ph+ leukemias in that it allows e.g. the detection of drug-resistant clones before clinical relapse of the patient.
DEFINITIONS
[0006] Within the context of this disclosure the following expressions, terms and abbreviations have the meanings as defined below:
[0007] In the expression "a mutated functional Abl kinase domain", the part "mutated Abl kinase domain" refers to the native human Abl kinase domain containing mutations including amino acid exchanges, amino acid deletions and/or amino acid additions.
[0008] In the expression "a mutated functional Abl kinase domain", the term "functional" indicates that the respective kinase domain possesses tyrosine kinase activity. Preferably, the kinase activity of the mutated functional Abl kinase domain is in the range of that of the native human Abl kinase domain.
[0009] In the expression "a mutated functional Abl kinase domain being resistant to inhibition of its tyrosine kinase activity by STI571 or a salt thereof", the term "resistant" means that STI571 inhibits the respective mutated functional Abl kinase domain with an IC50 that is higher than that of the native human Abl kinase domain, i.e. higher than about 0.025 μM, preferably higher than about 0.15 μM, more preferably higher than about 0.25 μM, most preferably higher than about 5 μM.
[0010] In the expression "amino acid sequence of the native human Abl kinase domain or an essentially similar sequence thereof", the part "or an essentially similar sequence thereof" refers to the amino acid sequence of the native human Abl kinase domain containing mutations, including amino acid exchanges, amino add deletions and/or amino acid additions, that are not essential for the functionality of the kinase and its resistance to inhibition by STI571 or a salt thereof within the meaning of the term "functional" and "resistant" as defined hereinabove.
[0011] The expression "replaced by another amino acid" refers to the replacement of a certain natural amino acid by another natural amino acid.
[0012] The names of the amino acids are either written out or the one letter or three letter codes are used. Mutations are referred to by accepted nomenclature, e.g. "Ala380Thr" or "380 Ala→Thr" both indicating that alanine at position 380 is replaced by threonine.
[0013] SEQ ID NO:1 represents the cDNA coding for the native human Abl protein (human c-abl mRNA; GenBank Accession No.: X16416).
[0014] SEQ ID NO:2 represents the amino acid sequence of the native human Abl protein (human c-Abl; SwissProt Acc. No.: P00519).
[0015] Unless indicated otherwise, the number given for a certain amino acid refers to the numbering of the amino acids in SEQ ID NO:2. In an amino acid sequence that is essentially similar to the amino acid sequence of the native human Abl kinase domain within the meaning as defined above, the amino acids are numbered in accordance with the numbering of the amino acids in SEQ ID NO:2.
[0016] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
[0017] A "host cell", refers to a prokaryotic or eukaryotic cell that contains heterologous DNA that has been introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, and the like.
DESCRIPTION OF THE INVENTION
[0018] In practicing the present invention, many conventional techniques in molecular biology, microbiology, and recombinant DNA are used. These techniques are well known and are explained in, for example, Current Protocols in Molecular Biology, Volumes I, II, and III, 1997 (F. M. Ausubel ed.); Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D. N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.); Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription and Translation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986 (R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press); Perbal, 1984, A Practical Guide to Molecular Cloning; the series, Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds., respectively).
[0019] In particular, the polypeptides of the present invention can be produced by recombinant DNA technology using techniques well-known in the art. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the sequences encoding the polypeptides of the invention and appropriate transcriptional/translational control signals. A variety of host-expression vector systems can be utilized to express the polypeptides of the invention.
(1). The invention relates to an isolated polypeptide which comprises a mutated functional Abl kinase domain that is resistant to inhibition of its tyrosine kinase activity by N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi- perazin-1-ylmethyl)-benzamide or a salt thereof. (2) The invention further relates in particular to an isolated polypeptide which comprises a mutated functional Abl kinase domain comprising the amino acid sequence of the native human Abl kinase domain or an essentially similar sequence thereof in which at least one amino acid is replaced by another amino acid, said mutated functional Abl kinase domain being resistant to inhibition of its tyrosine kinase activity by N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi- perazin-1-ylmethyl)-benzamide or a salt thereof. (3) The invention especially relates to an isolated polypeptide which comprises a mutated functional Abl kinase domain comprising the amino acid sequence of the native human Abl kinase domain or an essentially similar sequence thereof in which at least one amino acid selected from Leu248, Glu255, Lys271, Glu286, Met290, Thr315, Tyr320, Asn322, Glu373, His 375 and Ala380 is replaced by another amino acid, said mutated functional Abl kinase domain being resistant to inhibition of its tyrosine kinase activity by N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi- perazin-1-ylmethyl)-benzamide or a salt thereof. (4) A preferred embodiment of the invention relates to an isolated polypeptide which comprises a mutated functional Abl kinase domain comprising the amino acid sequence of the native human Abl kinase domain or an essentially similar sequence thereof in which at least one amino acid selected from Leu248, Glu255, Lys271, Glu286, Met290, Tyr320, Asn322, Glu373, His375 and Ala380 is replaced by another amino acid, said mutated functional Abl kinase domain being resistant to inhibition of its tyrosine kinase activity by N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi- perazin-1-ylmethyl)-benzamide or a salt thereof. (5) Another preferred embodiment of the invention relates to an isolated polypeptide which comprises a mutated functional Abl kinase domain comprising the amino acid sequence of the native human Abl kinase domain or an essentially similar sequence thereof in which at least one amino acid selected from Leu248, Lys271, Glu286, Met290, Tyr320, Asn322, Glu373, His375 and Ala380 is replaced by another amino acid, said mutated functional. Abl kinase domain being resistant to inhibition of its tyrosine kinase activity by N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi- perazin-1-ylmethyl)-benzamide or a salt thereof. (6) Another especially preferred embodiment of the invention relates to an isolated polypeptide which comprises a mutated functional Abl kinase domain comprising the amino acid-sequence of the native human Abl kinase domain or an essentially similar sequence thereof in which at least one amino acid selected from Glu255, Thr315 and Ala380 is replaced by another amino acid, said mutated functional Abl kinase domain being resistant to inhibition of its tyrosine kinase activity by N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi- perazin-1-ylmethyl)-benzamide or a salt thereof. (7) Another very preferred embodiment of the invention relates to an isolated polypeptide which comprises a mutated functional Abl kinase domain comprising the amino acid sequence of the native human Abl kinase domain or an essentially similar sequence thereof in which at least one amino acid selected from Glu255 and Ala380 is replaced by another amino acid, said mutated functional Abl kinase domain being resistant to inhibition of its tyrosine kinase activity by N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi- perazin-1-ylmethyl)-benzamide or a salt thereof. (8) Most preferably the invention relates to an isolated polypeptide according to any one of the preceding paragraphs (2)-(7), wherein in the amino acid sequence of the native human Abl kinase domain or an essentially similar sequence thereof a single amino acid is replaced by another amino acid. (9) The invention relates very especially preferred to an isolated polypeptide which comprises a mutated functional Abl kinase domain comprising the amino acid sequence of the native human Abl kinase domain or an essentially similar sequence thereof in which Glu255 is replaced by another amino acid, said mutated functional Abl kinase domain being resistant to inhibition of its tyrosine kinase activity by N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi- perazin-1-ylmethyl)-benzamide or a salt thereof. (10) Most especially preferred the invention relates to an isolated polypeptide which comprises a mutated functional Abl kinase domain comprising the amino acid sequence of the native human Abl kinase domain or an essentially similar sequence thereof that contains at least one amino acid mutation selected from Glu255Val, Glu255Lys, Thr315Val, Thr315Leu, Thr315Met, Thr315Gln, Thr315Phe and Ala380Thr, said mutated functional Abl kinase domain being resistant to inhibition of its tyrosine kinase activity by N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi- perazin-1-ylmethyl)-benzamide or a salt thereof. (11) In a further very preferred embodiment the invention relates to an isolated polypeptide which comprises a mutated functional Abl kinase domain comprising the amino acid sequence of the native human Abl kinase domain or an essentially similar sequence thereof that contains at least one amino acid mutation selected from Glu255Val, Thr315Val, Thr315Leu, Thr315Met, Thr315Gln, Thr315Phe and Ala380Thr, said mutated functional Abl kinase-domain being resistant to inhibition of its tyrosine kinase activity by N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi- perazin-1-ylmethyl)benzamide or a salt thereof. (12) In another especially preferred embodiment the invention relates to an isolated polypeptide which comprises a mutated functional Abl kinase domain comprising the amino add sequence of the native human Abl kinase domain or an essentially similar sequence thereof that contains at least one amino add mutation selected from Thr315Leu, Thr315Met, Thr315Gln and Thr315Phe, said mutated functional Abl kinase domain being resistant to inhibition of its tyrosine kinase activity by N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi- perazin-1-ylmethyl)-benzamide or a salt thereof. (13) Most preferably the invention relates to an isolated polypeptide according to any one of the preceding paragraphs (10)-(12), wherein the amino acid sequence of the native human Abl kinase domain or an essentially similar sequence thereof contains a single amino acid mutation. (14) Preferred above all the invention relates to an isolated polypeptide which comprises a mutated functional Abl kinase domain comprising the amino acid sequence of the native human Abl kinase domain or an essentially similar sequence thereof that contains the amino acid mutation Glu255Val, said mutated functional Abl kinase domain being resistant to inhibition of its tyrosine kinase activity by N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi- perazin-1-ylmethyl)-benzamide or a salt thereof. (15) In a preferred embodiment the invention relates to an isolated polypeptide according to any one of the preceding paragraphs (2)-(14), wherein the amino acid sequence of the native human Abl kinase domain consists of amino acids 229-500 of SEQ ID NO:2. (16) In another preferred embodiment the invention relates to an isolated polypeptide according to any one of the preceding paragraphs (2)-(15), said isolated polypeptide being a Bcr-Abl tyrosine kinase. (17) In yet another preferred embodiment the invention relates to the use of an isolated polypeptide of any one of the preceding paragraphs (2) to (16) to screen for compounds which inhibit the tyrosine kinase activity of said polypeptide. (18) The invention also relates to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide according to any one of the preceding paragraphs (2)-(16). (19) The invention further relates to the use of a nucleic acid molecule of the preceding paragraph (18) in the production of a polypeptide of any one of the preceding paragraphs (2) to (16) for use in screening for compounds which inhibit the tyrosine kinase activity of said polypeptide. (20) The invention also relates to a recombinant vector comprising a nucleic acid molecule according to the preceding paragraph (18). (21) The invention further relates especially to a recombinant vector according to the preceding paragraph (20), which is a recombinant expression vector. (22) The invention also relates to a host cell comprising a recombinant vector according to the preceding paragraph (20) or (21).
[0020] Preferably the invention relates to an isolated polypeptide which comprises a mutated functional Abl kinase domain comprising the amino acid sequence of the native human Abl kinase domain in which at least one amino acid is replaced by another amino acid, said mutated functional Abl kinase domain being resistant to inhibition of its tyrosine kinase activity by N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi- perazin-1-ylmethyl)-benzamide or a salt thereof.
[0021] Most preferred are the mutations described herein, which are present in patients who suffer from Philadelphia chromosome-positive leukemia and are resistant against treatment with STI571.
[0022] A preferred salt of STI571 is the methane sulfonate salt described in WO 99/03854.
[0023] Screening for compounds which inhibit the tyrosine kinase activity of the polypeptides of the invention may be done for example by using an isolated polypeptide of the invention in any in vitro tyrosine kinase phosphorylation assay known in the art and determining the potential of a compound to inhibit the tyrosine kinase activity of a polypeptide of the invention in such an assay.
[0024] High-throughput screening assays known in the art may be used to screen large compound libraries for compounds which inhibit the tyrosine kinase activity of the polypeptides of the invention.
[0025] Besides the random screening of large compound libraries, the polypeptides of the present invention may also be used in the following screening approach: The 3-dimensional structure of a polypeptide of the invention is determined by e.g. X-ray crystallography. The atomic coordinates of a polypeptide of the invention are then used to design a potential inhibitor. Said potential inhibitor is then synthesized and tested for its ability to inhibit the tyrosine kinase activity of the polypeptide of the invention in any in vitro tyrosine kinase phosphorylation assay.
EXAMPLES
[0026] The following Examples serve to illustrate the invention without limiting its scope.
Example 1
Methods:
Plasmids and Site Directed Mutagenesis:
[0027] The hybrid cDNA coding for HckAblSH1 was cloned by amplifying the respective DNA fragments from pUCΔNdeI/XbaIHck [Warmuth M. et al., J. Biol. Chem. 272, 33260-70 (1997)] and pcDNA3bcr-abl. These fragments were ligated blunt end to yield pUCΔNdeI/XbaIhckablSH1. Because of its relatively small size when compared to Bcr-Abl or c-Abl, this construct, hckAblSH1, allowed to introduce point mutations into the kinase domain of Abl by a one step cloning procedure. Point mutations were introduced into hckablSH1 using the QUICKCHANGE® site directed mutagenesis protocol from Stratagene (La Jolla, Calif.). In order to introduce point mutations into Bcr-Abl, a KpnI/Eco47III-subfragment of Bcr-Abl containing the sequence coding for Bcr-Abl's kinase domain was cloned into pUCΔNdeI/XbaI engineered by site directed mutagenesis to contain an Eco47111 site in the polylinker. After introduction of point mutations, this fragment was first recloned into pcDNA3abl. Thereafter, the 5' part of abl up to the KpnI site was substituted by Bcr coding sequences using a KpnI-fragment from pcDNA3bcr-abl. All mutations were confirmed by sequencing. For expression in Cos7 and 32D cells cDNAs were cloned into pApuro.
Cell Lines:
[0028] Parental 32D cells as well as 32D cells expressing Bcr-Abl and mutants thereof (32Dp210) were grown in Iscove's modified dulbeccos media (IMDM) supplemented with 10% FBS. COST cells were cultured in Dulbecco's modified eagle medium (DMEM) containing 4.5 g/l glucose) and supplemented with 10% FBS. All media and FBS were purchased from Gibco Life Technologies, Inc, Karlsruhe, Germany.
Transfection of Cells:
[0029] Cos7 cells were transfected using EFFECTEN® transfection reagent as to the guidelines of the manufacturer (Quiagen, Hilden, Germany). 32D cells were transfected by electroporation. Puromycin was used for selection at a concentration of 1 μg/ml.
Cell Lysis:
[0030] Cos7 cells were lysed as described recently [Warmuth M. et al., J. Biol. Chem. 272, 33260-70 (1997)]. For lysis, exponentially growing 32D cells were harvested and washed twice in cold PBS. For experiments evaluating the activity profile of STI571, cells were incubated with the indicated concentrations of inhibitor or with DMSO at a density of 5×106 cells/ml for 1.5-2 h. 107 cells were lysed in 100 μl of lysis buffer containing 1% NP-40, 20 mM Tris (pH 8.0), 50 mM NaCl, and 10 mM EDTA, 1 mM phenylmethylsulfonylfluorid, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 2 mM sodium orthovanadate. After resuspension in lysis buffer, cells were incubated for 25 min on ice. Finally, unsoluble material was removed by centrifugation at 15,000 g. Clarified lysates were checked for protein concentrations using a BioRad protein assay.
Immunoprecipitation:
[0031] For immunoprecipitation 150 μl of standardized 32D cell lysate was diluted by addition of 450 μl of incubation buffer containing 20 mM Tris (pH 8.0), 50 mM NaCl, and 10 mM EDTA, 1 mM phenylmethylsulfonylfluorid, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 2 mM sodium orthovanadate and incubated with 5 μg of the indicated antibodies for 2 hours on a overhead rotor at 4° C. Sepharose A beads (Pharmacia Biotech Inc., Freiburg, Germany) were prepared by washing twice in IP buffer [0.1% NP-40, 20 mM Tris (pH 8.0), 50 mM NaCl, and 10 mM EDTA, 1 mM phenylmethylsulfonylfluorid, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 2 mM sodium orthovanadate] and added to each sample for 2 additional hours. Finally, the immunoprecipitates were washed three times in IP buffer, subsequently boiled in 2× sample buffer and prepared for SDS-PAGE.
Gel Electrophoresis and Immunoblotting:
[0032] Gel electrophoresis and immunoblotting were performed as described [Danhauser-Riedl S. et al., Cancer Res. 56, 3589-96 (1996); Warmuth M. et al., J. Biol. Chem. 272, 33260-70 (1997)] with some minor modifications. Proteins were routinely blotted on nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Membranes were blocked in 5% milk powder for 1 h. Primary and secondary antibodies were diluted-1:500 to 1:5000 in TBS containing 1% milk powder. Proteins were detected using the ECL or ECL Plus detection system as recommended by the manufacturer (Amersham, Braunschweig, Germany).
Detection of Apoptosis by Flow Cytometry:
[0033] For assessing apoptosis induced by the various kinase inhibitors, cells were incubated with the indicated concentrations of STI571 at a density of 5×104 per ml. Apoptosis was routinely assessed by measuring the binding of FITC-conjugated Annexin V to the membranes of apoptosing cells. About 5×104 cells were taken at the indicated time points and washed once in PBS. Thereafter, cells were resuspended in 195 μl of Annexin V binding buffer and 5 μl of Annexin V-FITC (Bender MedSystems Diagnostics, Vienna, Austria) were added. Cells were mixed and incubated at room temperature for 10-20 min. Afterwards, cells were pelleted again, washed once and resuspended in 190 μl of Annexin V binding buffer. 10 μl of a 20 μg/ml propidium iodide stock solution were added and the ratio of Annexin V-positive to negative cells was determined by FACS-analysis using a Coulter EPICS XL 4-color cytometer.
Results:
[0034] Mutations to either Valine (V), Leucine (L), Isoleucine (I), Methionine (M), Glutamine (Q) or Phenylalanine (F) at position 315 and to either Serine (S), Cysteine (C) or Threonine (T) at position 380 were generated in a hybrid kinase, HckAblSH1, consisting of the SH2 and SH3 domain of Hck and the SH1/kinase domain of Abl. When expressed in Cos7 cells, these hybrid kinases and all mutants at position 315 and 380 showed a high spontaneous kinase activity, proving that these positions are not critical for ATP binding (Table 1). In marked contrast, when tested for inhibition by STI571, no inhibition was seen with up to 10 μM of compound for the mutants T315L, T315I, T315M, T315Q and T315F (Table 1), whereas HckAblSH1 wild-type (wt) could be inhibited with similar kinetics by STI571 as were found for Bcr-Abl (IC50 cellular tyrosine phosphorylation (IC50CTP) approx. 0.5 μM). The mutants T315V and A380T retained some partial sensitivity but IC50CTP values were still higher than 10 μM. In contrast, the mutants A380S and A380C displayed sensitivity to STI571, which was comparable to HckAblSH1 wt (see Table 1 for summary).
TABLE-US-00001 TABLE 1 Influence of mutations of T315 and A380 of HckAblSH1 on kinase activity and inhibition by STI571 kinase activity Inhibition by STI571 T315V +++ IC50 > 10 T315L +++ CR T315I +++ CR T315M ++++ CR T315Q ++ CR T315F ++ CR A380C +++ NS A380S +++ NS A380T + IC50 > 10
All data based on inhibition of cellular tyrosine phosphorylation of transiently transfected Cos7 cells determined by Western blot analysis using the monoclonal α-phosphotyrosine antibody PY99. IC50 values were determined using scion image software. Complete remission (CR) was defined as no detectable reduction of cellular tyrosine phosphorylation by 10 μM STI571. NS (normal sensitivity)=inhibition with similar kinetics as HckAblSH1 wt.
[0035] Our data identify positions 315 and 380 as critical gatekeepers for the binding pocket of STI571, which contribute to define the sensitivity of individual protein kinases towards STI571. For example, the STI571-insensitive receptor tyrosine kinase Flt-3, which has high homology to the c-Kit and the PDGF-R kinases, has a phenylalanine at the position homologous to T315, which would, based on our data, not be in accordance with STI571 binding. In a similar way, the resistance of most other kinases tested with STI571 could be explained.
[0036] In order to investigate whether and to what degree some of the above described point mutations of the gatekeeper position T315 are able to induce biological resistance towards STI571 we introduced into full length Bcr-Abl the mutations T315V, T315L, T315I, T315M, T315Q and T315F. When expressed in Cos7 cells all mutants displayed kinase activity close to or similar to wild-type Bcr-Abl (Bcr-ABLwt). If tested for inhibition by STI571, identical results were obtained as described for the corresponding mutations in HckAblSH1 (Table 2). Similar to Scr-Ablwt, expression of these mutants in 32D, an IL-3-dependent, hematopoietic cell line of murine origin, gave rise to cell lines growing IL-3 independently. Exposure of 32 DBcr-Ablwt cells to 1 or 10 μM STI571 lead to a rapid stop of proliferation and induction of apoptotic cell death in more than 90% of cells. On the contrary, if T315 mutant Bcr-Abl kinases, for example T315I, were expressed the block in proliferation and the induction of apoptosis caused by 1 μM STI571 were completely abolished (Table 2) and the effects of STI571 seen at 10 μM were reduced to levels found in control experiments using parental 32D cells grown in the presence of IL-3. Phosphotyrosine blots of samples of cells expressing either wt or mutant Bcr-Abl proteins confirmed that mutations at position 315 completely abolished the effect of STI571 on Abl auto- and substrate phosphorylation, with the exception of T315V which was still to some degree inhibited by STI571 but displayed a similar biological phenotype as the other mutants (Table 2). This suggests that the reminder biological activity of STI571 at 10 μM was rather due to cytotoxicity than to a reminder sensitivity of the mutants or cross-reaction of STI571 with another tyrosine kinase. In summary, all mutations lifted the IC50 for inhibition of proliferation (IC50IOP) from 0.09 to approximately 7.5 μM and for inhibition of survival (IC50IOS) from 0.5 to more than 10 μM (Table 2). Taken together, these data show that mutations of "molecular gatekeeper" positions as described above are able to confer complete biological resistance towards STI571 in a cell culture model.
TABLE-US-00002 TABLE 2 Biochemical and biological characterization of mutations of T315 in Bcr-Abl to amino acids with longer side chains IC50 (μM) cellular Induction kinase of growth tyrosine factor- IC50 phos- independent (μM) IC50 (μM) kinase phorylation growth in apoptosis proliferation mutant activity Cos7 32D 32D 32D 32D (32D) >10 7.5 wt +++ 0.25 0.25 yes 0.5 0.09 T315V +++ >10 >10 yes >10 7.5 T315L ++++ c.r. c.r. yes >10 7.5 T315I +++ c.r. c.r. yes >10 7.5 T315M ++++ c.r. c.r. yes >10 7.5 T315Q +++ c.r. c.r. yes >10 7.5 T315F +++ c.r. c.r. yes >10 7.5 c.r: complete remission (no detectable reduction of cellular tyrosine phosphrylation by 10 μM TI571)
Example 2
[0037] STI571 inhibits the Abl tyrosine kinase with an IC50 of 0.025 μM for purified Bcr-Abl and c-Abl but not the fms or the Src family kinases. The mechanism of inhibition is through competitive inhibition of ATP binding. To better understand the mechanism of specificity of the tyrosine kinase inhibitor the Abl kinase was compared to a model of the Lck kinase domain. This model predicts the following sites are critical for STI571 association: L248, Y320, N322, E373, H375 and A380. Each of these residues were changed to the corresponding residue in Src or fms and IC50 values for STI571 with each mutant were determined. L248A and H375L yielded kinase inactive mutants, Y320K, N322S, E373N and A380G had IC50 values identical to wild type Abl. A380T, however, demonstrated an IC50 of 0.34 μM suggesting that STI571 bound less efficiently when a larger residue replaced the alanine. Recent crystallization of the Abl kinase domain with a related inhibitor shows that the configuration of the activation loop of the Abl kinase domain differs significantly from that of the Src family kinases. This structure identifies K271, E286, M290, T315, M318 and D381 as critical contacts of STI571. All of these residues are conserved between Src and Abl. The last two of these bind STI571 via their peptide backbone, thus mutants in these residues cannot be created. The remainder of the residues were mutated to residues lacking the potential for hydrogen bonding and IC50 values were determined. K271R, E286L and M290A were kinase inactive. T315V had an IC50 value of 0.35 μM, which is consistent with the crystal structure of the Abl kinase domain which predicts that the side chain of T315 forms a critical hydrogen bond with STI571.
Example 3
[0038] A group of 32 patients who are either refractory to treatment with STI571 or who relapsed whilst being treated were investigated. The median duration of therapy was 95 days; prior to STI571 treatment, two patients were in chronic phase, nine in accelerated phase, 20 in myeloid and, and one in lymphoid blast crisis of the disease. Reverse transcriptase-polymerase chain reaction (RT-PCR) products specific for the Bcr-Abl tyrosine kinase domain were sequenced
[0039] (Heminested RT-PCR was performed to amplify the sequence specifically coding for the Bcr-Abl tyrosine kinase:
TABLE-US-00003 1st step B2B ACAGAATTCCGCTGACCATCAATAAG and A7- AGACGTCGGACTTGATGGAGAACT; 2nd step FA4+ AAGCGCAACAAGCCCACTGTCTAT and A7-).
[0040] An acquired A→T point mutation at position 58802 (GeneBank accession number U07563, locus HSABLGR3)--which results in a Glu255Val substitution--was detected in one patient. Restriction analysis of cDNA and genomic DNA (RT-PCR and genomic PCR were performed using primers A4+ TCACCACGCTCCATTATCCA, A4-CTTCCACACGCTCTCGTACA; Mnl I restriction digest of PCR products; removal of an Mnl I restriction site as the result of the point mutation A58802T) was used to confirm the presence of the mutation and to track it during the course of treatment. Only wild-type Abl sequence was present before the STI571 therapy. The patient was treated with STI571 in late chronic phase, went into complete hematologic remission, but progressed to blast crisis after five months. Reactivation of Bcr-Abl was confirmed by Crk1 immunoblotting [K. Senechal, Mol. Cell. Biol. 18, 5082 (1998)]. The relative proportion of phosphorylated Crk1 (reflecting active Bcr-Abl) was 49% before STI571 therapy, 24% at day 27, 28% at day 83, and 77% at the time of clinical resistance at day 166. The biological significance of the Glu255Val change is determined by an Abl autophosphorylation assay. STI571 inhibits wild-type Abl with an IC50 of 0.025 μM. The mutation leads to a virtual insensitivity to STI571, with an IC50 of >5 μM.
Example 4
[0041] The Bcr-Abl kinase domain from cells obtained from 12 CML and Ph+ acute leukemia patients who relapsed while receiving STI571 was sequenced. A functional point-mutation in the kinase domain in one case was identified. This was a G→A change that results in a Glu→Lys substitution at position 255 of Abl.
Example 5
Patients and Sample Preparation:
[0042] Thirty bone marrow samples from 21 patients with Ph+ALL who were enrolled into consecutive "Phase II study to determine the safety and anti-leukemic effect of STI571 in adult patients with Ph+ acute leukemias" were analyzed. According to the study protocol, these patients had relapsed ALL or were refractory after at least 2 cycles of standard chemotherapy. From all of the patients, samples were obtained before beginning STI571 treatment: 13 of these samples were from individuals who later were classified as good responders to STI571 (Nos. 1-13, sensitive, S) including 12 patients with hematological complete remission (CR) and one patient with partial remission (PR) but complete peripheral hematological recovery (No. 1). Eight samples were collected from individuals who later were found not to respond to STI571 (Nos. 14-21, primarily resistant, R) including 6 patients without any hematological response, one with cytoreduction in the bone marrow but persistent peripheral leukemic cells (No. 20) and another with PR but incomplete peripheral hematological recovery (No. 16). Matched bone marrow samples from 9 patients (Nos. 1-5 and Nos. 14-17) were also obtained while they were on treatment with STI571. Mononuclear cells were separated by density gradient centrifugation through Ficoll-Hypaque (Biochrom, Berlin, Germany). Total RNA was extracted using the acid guanidium/phenol/chloroform method with minor modifications. [Puissant C. and Houdebine L. M., Biotechniques 8, 148-149 (1990)]. Only samples with leukemic blast cell infiltration of more than 80% were included into the analysis.
[0043] Reverse transcription polymerase chain reaction and sequencing analysis: One microgram of total RNA was used for reverse transcription (RT) by Superscript II RT (Life Technologies, Grand Island, N.Y.) according to standard protocols, Primers specific for the ATP binding site of ABL including the `loop` were designed using gene bank information GI6382056: ATP-F 5'-GCG CAA CAA GCC CAC TGT CT-3'; ATP-R 5'-GCA CTC CCT CAG GTA GTC CA-3' and LOOP-F 5'-TGG ACT ACC TGA GGG AGT GC-3'; LOOP-R 5'-CGG TAG TCC TTC TCT AGC AGC-3'. Oligonucleotides were synthesized by Life Technologies. Polymerase chain reaction (PCR) was performed as described previously [Hofmann W. K. et al., Leuk. Res. 25, 333-338 (2001)] using an annealing temperature of 58° C. PCR-products were separated on a 2% agarose gel containing 0.3 mg/ml ethidium bromide and purified using the QIAquick purification system (Qiagen, Valencia, Calif.) according to the manufacturer's protocol. The purified DNA was directly sequenced in both directions (sense and antisense) by the ABI PRISM dye terminator cycle sequencing reaction (Perkin-Elmer, Foster, Calif.).
Results:
[0044] Analysis of the sequence of the ATP binding site revealed a single point mutation at nucleotide 1127 (GI6382056) changing a G to an A resulting in a substitution at codon 255 of Lys (mutant) for a Glu (wild-type). This mutation was found in 6 samples from patients after they were treated with STI571 (Nos. 1, 2, 4, 5, 15, 16) but mutations were not found in any other sample including the matched samples from the patients before beginning treatment with STI571 (Table 3). The change was verified by sequencing from both the sense and antisense directions. In addition, one sample (No: 17) from a patient with an aberrant CALL had a single point mutation at nucleotide 1308 changing a C to T resulting in a substitution at codon 315 of isoleucine (mutant) for a threonine (wild-type). This sample was unusual because the cells also expressed CD33, a cell surface protein expressed on myeloid cells.
[0045] Our data strongly suggest that E255K developed during treatment with STI571. Our analysis of matched samples found, that none of the samples from untreated patients (including sensitive patients and those with primary resistance) had this mutation. In contrast, six of 9 samples (67%) from these patients undergoing treatment with STI571 had this substitution at E255. The overall frequency of mutations in the ATP binding site was 7 of 9 (78%) in our paired bone marrow samples from patients undergoing therapy with STI571.
TABLE-US-00004 TABLE 3 Matched bone marrow samples: Development of mutations in the Region coding for the ATP binding site of ABL during treatment of Ph+ ALL with STI571. ABS status ABS status prior to after treatment with Response to treatment with No. Diagnosis STI571 STI571 STI571 1 Ph+ cALL Wild type PR E255K 2 Ph+ cALL Wild type CR E255K 3 Ph+ cALL Wild type CR Wild type 4 Ph+ cALL Wild type CR E255K 5 Ph+ cALL Wild type CR E255K 14 Ph+ cALL Wild type no Wild type 15 Ph+ cALL Wild type no E255K 16 Ph+ pre B-ALL Wild type PR E255K 17 Ph+ cALL, Wild type no T315I CD33+ ABS, ATP binding site; PR, partial remission; CR, complete remission; Ph+ cALL, Philadelphia chromosome positive, common ALL (CD10+).
Sequence CWU
1
1
213393DNAHomo sapiensCDS(1)..(3393) 1atg ttg gag atc tgc ctg aag ctg gtg
ggc tgc aaa tcc aag aag ggg 48Met Leu Glu Ile Cys Leu Lys Leu Val
Gly Cys Lys Ser Lys Lys Gly 1 5
10 15 ctg tcc tcg tcc tcc agc tgt tat ctg
gaa gaa gcc ctt cag cgg cca 96Leu Ser Ser Ser Ser Ser Cys Tyr Leu
Glu Glu Ala Leu Gln Arg Pro 20 25
30 gta gca tct gac ttt gag cct cag ggt ctg
agt gaa gcc gct cgt tgg 144Val Ala Ser Asp Phe Glu Pro Gln Gly Leu
Ser Glu Ala Ala Arg Trp 35 40
45 aac tcc aag gaa aac ctt ctc gct gga ccc agt
gaa aat gac ccc aac 192Asn Ser Lys Glu Asn Leu Leu Ala Gly Pro Ser
Glu Asn Asp Pro Asn 50 55
60 ctt ttc gtt gca ctg tat gat ttt gtg gcc agt
gga gat aac act cta 240Leu Phe Val Ala Leu Tyr Asp Phe Val Ala Ser
Gly Asp Asn Thr Leu 65 70 75
80 agc ata act aaa ggt gaa aag ctc cgg gtc tta ggc
tat aat cac aat 288Ser Ile Thr Lys Gly Glu Lys Leu Arg Val Leu Gly
Tyr Asn His Asn 85 90
95 ggg gaa tgg tgt gaa gcc caa acc aaa aat ggc caa ggc
tgg gtc cca 336Gly Glu Trp Cys Glu Ala Gln Thr Lys Asn Gly Gln Gly
Trp Val Pro 100 105
110 agc aac tac atc acg cca gtc aac agt ctg gag aaa cac
tcc tgg tac 384Ser Asn Tyr Ile Thr Pro Val Asn Ser Leu Glu Lys His
Ser Trp Tyr 115 120 125
cat ggg cct gtg tcc cgc aat gcc gct gag tat ctg ctg agc
agc ggg 432His Gly Pro Val Ser Arg Asn Ala Ala Glu Tyr Leu Leu Ser
Ser Gly 130 135 140
atc aat ggc agc ttc ttg gtg cgt gag agt gag agc agt cct ggc
cag 480Ile Asn Gly Ser Phe Leu Val Arg Glu Ser Glu Ser Ser Pro Gly
Gln 145 150 155
160 agg tcc atc tcg ctg aga tac gaa ggg agg gtg tac cat tac agg
atc 528Arg Ser Ile Ser Leu Arg Tyr Glu Gly Arg Val Tyr His Tyr Arg
Ile 165 170 175
aac act gct tct gat ggc aag ctc tac gtc tcc tcc gag agc cgc ttc
576Asn Thr Ala Ser Asp Gly Lys Leu Tyr Val Ser Ser Glu Ser Arg Phe
180 185 190
aac acc ctg gcc gag ttg gtt cat cat cat tca acg gtg gcc gac ggg
624Asn Thr Leu Ala Glu Leu Val His His His Ser Thr Val Ala Asp Gly
195 200 205
ctc atc acc acg ctc cat tat cca gcc cca aag cgc aac aag ccc act
672Leu Ile Thr Thr Leu His Tyr Pro Ala Pro Lys Arg Asn Lys Pro Thr
210 215 220
gtc tat ggt gtg tcc ccc aac tac gac aag tgg gag atg gaa cgc acg
720Val Tyr Gly Val Ser Pro Asn Tyr Asp Lys Trp Glu Met Glu Arg Thr
225 230 235 240
gac atc acc atg aag cac aag ctg ggc ggg ggc cag tac ggg gag gtg
768Asp Ile Thr Met Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Glu Val
245 250 255
tac gag ggc gtg tgg aag aaa tac agc ctg acg gtg gcc gtg aag acc
816Tyr Glu Gly Val Trp Lys Lys Tyr Ser Leu Thr Val Ala Val Lys Thr
260 265 270
ttg aag gag gac acc atg gag gtg gaa gag ttc ttg aaa gaa gct gca
864Leu Lys Glu Asp Thr Met Glu Val Glu Glu Phe Leu Lys Glu Ala Ala
275 280 285
gtc atg aaa gag atc aaa cac cct aac ctg gtg cag ctc ctt ggg gtc
912Val Met Lys Glu Ile Lys His Pro Asn Leu Val Gln Leu Leu Gly Val
290 295 300
tgc acc cgg gag ccc ccg ttc tat atc atc act gag ttc atg acc tac
960Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Thr Glu Phe Met Thr Tyr
305 310 315 320
ggg aac ctc ctg gac tac ctg agg gag tgc aac cgg cag gag gtg aac
1008Gly Asn Leu Leu Asp Tyr Leu Arg Glu Cys Asn Arg Gln Glu Val Asn
325 330 335
gcc gtg gtg ctg ctg tac atg gcc act cag atc tcg tca gcc atg gag
1056Ala Val Val Leu Leu Tyr Met Ala Thr Gln Ile Ser Ser Ala Met Glu
340 345 350
tac ctg gag aag aaa aac ttc atc cac aga gat ctt gct gcc cga aac
1104Tyr Leu Glu Lys Lys Asn Phe Ile His Arg Asp Leu Ala Ala Arg Asn
355 360 365
tgc ctg gta ggg gag aac cac ttg gtg aag gta gct gat ttt ggc ctg
1152Cys Leu Val Gly Glu Asn His Leu Val Lys Val Ala Asp Phe Gly Leu
370 375 380
agc agg ttg atg aca ggg gac acc tac aca gcc cat gct gga gcc aag
1200Ser Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala His Ala Gly Ala Lys
385 390 395 400
ttc ccc atc aaa tgg act gca ccc gag agc ctg gcc tac aac aag ttc
1248Phe Pro Ile Lys Trp Thr Ala Pro Glu Ser Leu Ala Tyr Asn Lys Phe
405 410 415
tcc atc aag tcc gac gtc tgg gca ttt gga gta ttg ctt tgg gaa att
1296Ser Ile Lys Ser Asp Val Trp Ala Phe Gly Val Leu Leu Trp Glu Ile
420 425 430
gct acc tat ggc atg tcc cct tac ccg gga att gac ctg tcc cag gtg
1344Ala Thr Tyr Gly Met Ser Pro Tyr Pro Gly Ile Asp Leu Ser Gln Val
435 440 445
tat gag ctg cta gag aag gac tac cgc atg gag cgc cca gaa ggc tgc
1392Tyr Glu Leu Leu Glu Lys Asp Tyr Arg Met Glu Arg Pro Glu Gly Cys
450 455 460
cca gag aag gtc tat gaa ctc atg cga gca tgt tgg cag tgg aat ccc
1440Pro Glu Lys Val Tyr Glu Leu Met Arg Ala Cys Trp Gln Trp Asn Pro
465 470 475 480
tct gac cgg ccc tcc ttt gct gaa atc cac caa gcc ttt gaa aca atg
1488Ser Asp Arg Pro Ser Phe Ala Glu Ile His Gln Ala Phe Glu Thr Met
485 490 495
ttc cag gaa tcc agt atc tca gac gaa gtg gaa aag gag ctg ggg aaa
1536Phe Gln Glu Ser Ser Ile Ser Asp Glu Val Glu Lys Glu Leu Gly Lys
500 505 510
caa ggc gtc cgt ggg gct gtg agt acc ttg ctg cag gcc cca gag ctg
1584Gln Gly Val Arg Gly Ala Val Ser Thr Leu Leu Gln Ala Pro Glu Leu
515 520 525
ccc acc aag acg agg acc tcc agg aga gct gca gag cac aga gac acc
1632Pro Thr Lys Thr Arg Thr Ser Arg Arg Ala Ala Glu His Arg Asp Thr
530 535 540
act gac gtg cct gag atg cct cac tcc aag ggc cag gga gag agc gat
1680Thr Asp Val Pro Glu Met Pro His Ser Lys Gly Gln Gly Glu Ser Asp
545 550 555 560
cct ctg gac cat gag cct gcc gtg tct cca ttg ctc cct cga aaa gag
1728Pro Leu Asp His Glu Pro Ala Val Ser Pro Leu Leu Pro Arg Lys Glu
565 570 575
cga ggt ccc ccg gag ggc ggc ctg aat gaa gat gag cgc ctt ctc ccc
1776Arg Gly Pro Pro Glu Gly Gly Leu Asn Glu Asp Glu Arg Leu Leu Pro
580 585 590
aaa gac aaa aag acc aac ttg ttc agc gcc ttg atc aag aag aag aag
1824Lys Asp Lys Lys Thr Asn Leu Phe Ser Ala Leu Ile Lys Lys Lys Lys
595 600 605
aag aca gcc cca acc cct ccc aaa cgc agc agc tcc ttc cgg gag atg
1872Lys Thr Ala Pro Thr Pro Pro Lys Arg Ser Ser Ser Phe Arg Glu Met
610 615 620
gac ggc cag ccg gag cgc aga ggg gcc ggc gag gaa gag ggc cga gac
1920Asp Gly Gln Pro Glu Arg Arg Gly Ala Gly Glu Glu Glu Gly Arg Asp
625 630 635 640
atc agc aac ggg gca ctg gct ttc acc ccc ttg gac aca gct gac cca
1968Ile Ser Asn Gly Ala Leu Ala Phe Thr Pro Leu Asp Thr Ala Asp Pro
645 650 655
gcc aag tcc cca aag ccc agc aat ggg gct ggg gtc ccc aat gga gcc
2016Ala Lys Ser Pro Lys Pro Ser Asn Gly Ala Gly Val Pro Asn Gly Ala
660 665 670
ctc cgg gag tcc ggg ggc tca ggc ttc cgg tct ccc cac ctg tgg aag
2064Leu Arg Glu Ser Gly Gly Ser Gly Phe Arg Ser Pro His Leu Trp Lys
675 680 685
aag tcc agc acg ctg acc agc agc cgc cta gcc acc ggc gag gag gag
2112Lys Ser Ser Thr Leu Thr Ser Ser Arg Leu Ala Thr Gly Glu Glu Glu
690 695 700
ggc ggt ggc agc tcc agc aag cgc ttc ctg cgc tct tgc tcc gcc tcc
2160Gly Gly Gly Ser Ser Ser Lys Arg Phe Leu Arg Ser Cys Ser Ala Ser
705 710 715 720
tgc gtt ccc cat ggg gcc aag gac acg gag tgg agg tca gtc acg ctg
2208Cys Val Pro His Gly Ala Lys Asp Thr Glu Trp Arg Ser Val Thr Leu
725 730 735
cct cgg gac ttg cag tcc acg gga aga cag ttt gac tcg tcc aca ttt
2256Pro Arg Asp Leu Gln Ser Thr Gly Arg Gln Phe Asp Ser Ser Thr Phe
740 745 750
gga ggg cac aaa agt gag aag ccg gct ctg cct cgg aag agg gca ggg
2304Gly Gly His Lys Ser Glu Lys Pro Ala Leu Pro Arg Lys Arg Ala Gly
755 760 765
gag aac agg tct gac cag gtg acc cga ggc aca gta acg cct ccc ccc
2352Glu Asn Arg Ser Asp Gln Val Thr Arg Gly Thr Val Thr Pro Pro Pro
770 775 780
agg ctg gtg aaa aag aat gag gaa gct gct gat gag gtc ttc aaa gac
2400Arg Leu Val Lys Lys Asn Glu Glu Ala Ala Asp Glu Val Phe Lys Asp
785 790 795 800
atc atg gag tcc agc ccg ggc tcc agc ccg ccc aac ctg act cca aaa
2448Ile Met Glu Ser Ser Pro Gly Ser Ser Pro Pro Asn Leu Thr Pro Lys
805 810 815
ccc ctc cgg cgg cag gtc acc gtg gcc cct gcc tcg ggc ctc ccc cac
2496Pro Leu Arg Arg Gln Val Thr Val Ala Pro Ala Ser Gly Leu Pro His
820 825 830
aag gaa gaa gct gaa aag ggc agt gcc tta ggg acc cct gct gca gct
2544Lys Glu Glu Ala Glu Lys Gly Ser Ala Leu Gly Thr Pro Ala Ala Ala
835 840 845
gag cca gtg acc ccc acc agc aaa gca ggc tca ggt gca cca ggg ggc
2592Glu Pro Val Thr Pro Thr Ser Lys Ala Gly Ser Gly Ala Pro Gly Gly
850 855 860
acc agc aag ggc ccc gcc gag gag tcc aga gtg agg agg cac aag cac
2640Thr Ser Lys Gly Pro Ala Glu Glu Ser Arg Val Arg Arg His Lys His
865 870 875 880
tcc tct gag tcg cca ggg agg gac aag ggg aaa ttg tcc agg ctc aaa
2688Ser Ser Glu Ser Pro Gly Arg Asp Lys Gly Lys Leu Ser Arg Leu Lys
885 890 895
cct gcc ccg ccg ccc cca cca gca gcc tct gca ggg aag gct gga gga
2736Pro Ala Pro Pro Pro Pro Pro Ala Ala Ser Ala Gly Lys Ala Gly Gly
900 905 910
aag ccc tcg cag agc ccg agc cag gag gcg gcc ggg gag gca gtc ctg
2784Lys Pro Ser Gln Ser Pro Ser Gln Glu Ala Ala Gly Glu Ala Val Leu
915 920 925
ggc gca aag aca aaa gcc acg agt ctg gtt gat gct gtg aac agt gac
2832Gly Ala Lys Thr Lys Ala Thr Ser Leu Val Asp Ala Val Asn Ser Asp
930 935 940
gct gcc aag ccc agc cag ccg gga gag ggc ctc aaa aag ccc gtg ctc
2880Ala Ala Lys Pro Ser Gln Pro Gly Glu Gly Leu Lys Lys Pro Val Leu
945 950 955 960
ccg gcc act cca aag cca cag tcc gcc aag ccg tcg ggg acc ccc atc
2928Pro Ala Thr Pro Lys Pro Gln Ser Ala Lys Pro Ser Gly Thr Pro Ile
965 970 975
agc cca gcc ccc gtt ccc tcc acg ttg cca tca gca tcc tcg gcc ctg
2976Ser Pro Ala Pro Val Pro Ser Thr Leu Pro Ser Ala Ser Ser Ala Leu
980 985 990
gca ggg gac cag ccg tct tcc act gcc ttc atc cct ctc ata tca acc
3024Ala Gly Asp Gln Pro Ser Ser Thr Ala Phe Ile Pro Leu Ile Ser Thr
995 1000 1005
cga gtg tct ctt cgg aaa acc cgc cag cct cca gag cgg atc gcc
3069Arg Val Ser Leu Arg Lys Thr Arg Gln Pro Pro Glu Arg Ile Ala
1010 1015 1020
agc ggc gcc atc acc aag ggc gtg gtc ctg gac agc acc gag gcg
3114Ser Gly Ala Ile Thr Lys Gly Val Val Leu Asp Ser Thr Glu Ala
1025 1030 1035
ctg tgc ctc gcc atc tct agg aac tcc gag cag atg gcc agc cac
3159Leu Cys Leu Ala Ile Ser Arg Asn Ser Glu Gln Met Ala Ser His
1040 1045 1050
agc gca gtg ctg gag gcc ggc aaa aac ctc tac acg ttc tgc gtg
3204Ser Ala Val Leu Glu Ala Gly Lys Asn Leu Tyr Thr Phe Cys Val
1055 1060 1065
agc tat gtg gat tcc atc cag caa atg agg aac aag ttt gcc ttc
3249Ser Tyr Val Asp Ser Ile Gln Gln Met Arg Asn Lys Phe Ala Phe
1070 1075 1080
cga gag gcc atc aac aaa ctg gag aat aat ctc cgg gag ctt cag
3294Arg Glu Ala Ile Asn Lys Leu Glu Asn Asn Leu Arg Glu Leu Gln
1085 1090 1095
atc tgc ccg gcg aca gca ggc agt ggt ccg gcg gcc act cag gac
3339Ile Cys Pro Ala Thr Ala Gly Ser Gly Pro Ala Ala Thr Gln Asp
1100 1105 1110
ttc agc aag ctc ctc agt tcg gtg aag gaa atc agt gac ata gtg
3384Phe Ser Lys Leu Leu Ser Ser Val Lys Glu Ile Ser Asp Ile Val
1115 1120 1125
cag agg tag
3393Gln Arg
1130
21130PRTHomo sapiens 2Met Leu Glu Ile Cys Leu Lys Leu Val Gly Cys Lys Ser
Lys Lys Gly 1 5 10 15
Leu Ser Ser Ser Ser Ser Cys Tyr Leu Glu Glu Ala Leu Gln Arg Pro
20 25 30 Val Ala Ser Asp
Phe Glu Pro Gln Gly Leu Ser Glu Ala Ala Arg Trp 35
40 45 Asn Ser Lys Glu Asn Leu Leu Ala
Gly Pro Ser Glu Asn Asp Pro Asn 50 55
60 Leu Phe Val Ala Leu Tyr Asp Phe Val Ala Ser Gly Asp
Asn Thr Leu 65 70 75
80 Ser Ile Thr Lys Gly Glu Lys Leu Arg Val Leu Gly Tyr Asn His Asn
85 90 95 Gly Glu Trp Cys
Glu Ala Gln Thr Lys Asn Gly Gln Gly Trp Val Pro 100
105 110 Ser Asn Tyr Ile Thr Pro Val Asn Ser
Leu Glu Lys His Ser Trp Tyr 115 120
125 His Gly Pro Val Ser Arg Asn Ala Ala Glu Tyr Leu Leu Ser
Ser Gly 130 135 140
Ile Asn Gly Ser Phe Leu Val Arg Glu Ser Glu Ser Ser Pro Gly Gln 145
150 155 160 Arg Ser Ile Ser Leu
Arg Tyr Glu Gly Arg Val Tyr His Tyr Arg Ile 165
170 175 Asn Thr Ala Ser Asp Gly Lys Leu Tyr Val
Ser Ser Glu Ser Arg Phe 180 185
190 Asn Thr Leu Ala Glu Leu Val His His His Ser Thr Val Ala Asp
Gly 195 200 205 Leu
Ile Thr Thr Leu His Tyr Pro Ala Pro Lys Arg Asn Lys Pro Thr 210
215 220 Val Tyr Gly Val Ser Pro
Asn Tyr Asp Lys Trp Glu Met Glu Arg Thr 225 230
235 240 Asp Ile Thr Met Lys His Lys Leu Gly Gly Gly
Gln Tyr Gly Glu Val 245 250
255 Tyr Glu Gly Val Trp Lys Lys Tyr Ser Leu Thr Val Ala Val Lys Thr
260 265 270 Leu Lys
Glu Asp Thr Met Glu Val Glu Glu Phe Leu Lys Glu Ala Ala 275
280 285 Val Met Lys Glu Ile Lys His
Pro Asn Leu Val Gln Leu Leu Gly Val 290 295
300 Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Thr
Glu Phe Met Thr Tyr 305 310 315
320 Gly Asn Leu Leu Asp Tyr Leu Arg Glu Cys Asn Arg Gln Glu Val Asn
325 330 335 Ala Val
Val Leu Leu Tyr Met Ala Thr Gln Ile Ser Ser Ala Met Glu 340
345 350 Tyr Leu Glu Lys Lys Asn Phe
Ile His Arg Asp Leu Ala Ala Arg Asn 355 360
365 Cys Leu Val Gly Glu Asn His Leu Val Lys Val Ala
Asp Phe Gly Leu 370 375 380
Ser Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala His Ala Gly Ala Lys 385
390 395 400 Phe Pro Ile
Lys Trp Thr Ala Pro Glu Ser Leu Ala Tyr Asn Lys Phe 405
410 415 Ser Ile Lys Ser Asp Val Trp Ala
Phe Gly Val Leu Leu Trp Glu Ile 420 425
430 Ala Thr Tyr Gly Met Ser Pro Tyr Pro Gly Ile Asp Leu
Ser Gln Val 435 440 445
Tyr Glu Leu Leu Glu Lys Asp Tyr Arg Met Glu Arg Pro Glu Gly Cys 450
455 460 Pro Glu Lys Val
Tyr Glu Leu Met Arg Ala Cys Trp Gln Trp Asn Pro 465 470
475 480 Ser Asp Arg Pro Ser Phe Ala Glu Ile
His Gln Ala Phe Glu Thr Met 485 490
495 Phe Gln Glu Ser Ser Ile Ser Asp Glu Val Glu Lys Glu Leu
Gly Lys 500 505 510
Gln Gly Val Arg Gly Ala Val Ser Thr Leu Leu Gln Ala Pro Glu Leu
515 520 525 Pro Thr Lys Thr
Arg Thr Ser Arg Arg Ala Ala Glu His Arg Asp Thr 530
535 540 Thr Asp Val Pro Glu Met Pro His
Ser Lys Gly Gln Gly Glu Ser Asp 545 550
555 560 Pro Leu Asp His Glu Pro Ala Val Ser Pro Leu Leu
Pro Arg Lys Glu 565 570
575 Arg Gly Pro Pro Glu Gly Gly Leu Asn Glu Asp Glu Arg Leu Leu Pro
580 585 590 Lys Asp Lys
Lys Thr Asn Leu Phe Ser Ala Leu Ile Lys Lys Lys Lys 595
600 605 Lys Thr Ala Pro Thr Pro Pro Lys
Arg Ser Ser Ser Phe Arg Glu Met 610 615
620 Asp Gly Gln Pro Glu Arg Arg Gly Ala Gly Glu Glu Glu
Gly Arg Asp 625 630 635
640 Ile Ser Asn Gly Ala Leu Ala Phe Thr Pro Leu Asp Thr Ala Asp Pro
645 650 655 Ala Lys Ser Pro
Lys Pro Ser Asn Gly Ala Gly Val Pro Asn Gly Ala 660
665 670 Leu Arg Glu Ser Gly Gly Ser Gly Phe
Arg Ser Pro His Leu Trp Lys 675 680
685 Lys Ser Ser Thr Leu Thr Ser Ser Arg Leu Ala Thr Gly Glu
Glu Glu 690 695 700
Gly Gly Gly Ser Ser Ser Lys Arg Phe Leu Arg Ser Cys Ser Ala Ser 705
710 715 720 Cys Val Pro His Gly
Ala Lys Asp Thr Glu Trp Arg Ser Val Thr Leu 725
730 735 Pro Arg Asp Leu Gln Ser Thr Gly Arg Gln
Phe Asp Ser Ser Thr Phe 740 745
750 Gly Gly His Lys Ser Glu Lys Pro Ala Leu Pro Arg Lys Arg Ala
Gly 755 760 765 Glu
Asn Arg Ser Asp Gln Val Thr Arg Gly Thr Val Thr Pro Pro Pro 770
775 780 Arg Leu Val Lys Lys Asn
Glu Glu Ala Ala Asp Glu Val Phe Lys Asp 785 790
795 800 Ile Met Glu Ser Ser Pro Gly Ser Ser Pro Pro
Asn Leu Thr Pro Lys 805 810
815 Pro Leu Arg Arg Gln Val Thr Val Ala Pro Ala Ser Gly Leu Pro His
820 825 830 Lys Glu
Glu Ala Glu Lys Gly Ser Ala Leu Gly Thr Pro Ala Ala Ala 835
840 845 Glu Pro Val Thr Pro Thr Ser
Lys Ala Gly Ser Gly Ala Pro Gly Gly 850 855
860 Thr Ser Lys Gly Pro Ala Glu Glu Ser Arg Val Arg
Arg His Lys His 865 870 875
880 Ser Ser Glu Ser Pro Gly Arg Asp Lys Gly Lys Leu Ser Arg Leu Lys
885 890 895 Pro Ala Pro
Pro Pro Pro Pro Ala Ala Ser Ala Gly Lys Ala Gly Gly 900
905 910 Lys Pro Ser Gln Ser Pro Ser Gln
Glu Ala Ala Gly Glu Ala Val Leu 915 920
925 Gly Ala Lys Thr Lys Ala Thr Ser Leu Val Asp Ala Val
Asn Ser Asp 930 935 940
Ala Ala Lys Pro Ser Gln Pro Gly Glu Gly Leu Lys Lys Pro Val Leu 945
950 955 960 Pro Ala Thr Pro
Lys Pro Gln Ser Ala Lys Pro Ser Gly Thr Pro Ile 965
970 975 Ser Pro Ala Pro Val Pro Ser Thr Leu
Pro Ser Ala Ser Ser Ala Leu 980 985
990 Ala Gly Asp Gln Pro Ser Ser Thr Ala Phe Ile Pro Leu
Ile Ser Thr 995 1000 1005
Arg Val Ser Leu Arg Lys Thr Arg Gln Pro Pro Glu Arg Ile Ala
1010 1015 1020 Ser Gly Ala
Ile Thr Lys Gly Val Val Leu Asp Ser Thr Glu Ala 1025
1030 1035 Leu Cys Leu Ala Ile Ser Arg Asn
Ser Glu Gln Met Ala Ser His 1040 1045
1050 Ser Ala Val Leu Glu Ala Gly Lys Asn Leu Tyr Thr Phe
Cys Val 1055 1060 1065
Ser Tyr Val Asp Ser Ile Gln Gln Met Arg Asn Lys Phe Ala Phe 1070
1075 1080 Arg Glu Ala Ile Asn
Lys Leu Glu Asn Asn Leu Arg Glu Leu Gln 1085 1090
1095 Ile Cys Pro Ala Thr Ala Gly Ser Gly Pro
Ala Ala Thr Gln Asp 1100 1105 1110
Phe Ser Lys Leu Leu Ser Ser Val Lys Glu Ile Ser Asp Ile Val
1115 1120 1125 Gln Arg
1130
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