WO1997039326A2 - Dosage in vitro a polarisation de fluorescence - Google Patents

Dosage in vitro a polarisation de fluorescence Download PDF

Info

Publication number
WO1997039326A2
WO1997039326A2 PCT/US1997/006746 US9706746W WO9739326A2 WO 1997039326 A2 WO1997039326 A2 WO 1997039326A2 US 9706746 W US9706746 W US 9706746W WO 9739326 A2 WO9739326 A2 WO 9739326A2
Authority
WO
WIPO (PCT)
Prior art keywords
protein
domains
proteins
test substance
domain
Prior art date
Application number
PCT/US1997/006746
Other languages
English (en)
Other versions
WO1997039326A3 (fr
Inventor
Berkley A. Lynch
Ian A. Macneil
Mark J. Zoller
Original Assignee
Ariad Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ariad Pharmaceuticals, Inc. filed Critical Ariad Pharmaceuticals, Inc.
Priority to JP09530451A priority Critical patent/JP2000512737A/ja
Priority to AU28076/97A priority patent/AU727108B2/en
Priority to EP97922393A priority patent/EP0898709A2/fr
Publication of WO1997039326A2 publication Critical patent/WO1997039326A2/fr
Publication of WO1997039326A3 publication Critical patent/WO1997039326A3/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

  • the invention relates to materials and methods for the identification of inhibitors of protein:protein interactions, especially those involved in cellular signal transduction.
  • Cellular signal transduction i.e., the series of events leading from extracellular events to intracellular sequelae, is an aspect of cellular function in both normal and disease states.
  • Numerous proteins that function as signal transducing molecules have been identified, including receptors, docking or recruiting proteins and enzymes such as receptor and non-receptor tyrosine kinases, phosphatases and other molecules with enzymatic or regulatory activities. These molecules generally demonstrate the capacity to associate specifically with other proteins to form a signaling complex that can alter cell activity.
  • Signaling proteins often contain domain(s) of conserved sequence, which serve as non-catalytic modules that direct protein-protein interactions during signal transduction.
  • domains include among others, SH2, phosphotyrosine interaction ("PI"), WW and SH3 domains.
  • SH2 and PI domains recognize, i.e., bind to, proteins containing characteristic peptide sequences which include one or more phosphorylated tyrosine residues.
  • WW and SH3 domains recognize proteins containing characteristic peptide sequences which need not contain phosphotyrosine residues.
  • compounds capable of interfering with that proteimprotein interaction may be useful in preventing or treating the disease or condition in mammals, including human patients.
  • receptor domains e.g., SH2 and PI domains
  • SH2 and PI domains phosphotyrosine-containing ligands
  • Binding assays have been described for detecting test substances which interfere with the association of proteins containing an SH2 domain with their phosphotyrosine containing ligands. See, e.g., Pawson, US Patent No. 5,352,660. More recently reported binding assays have utilized surface plasmon resonance (Biacore) [see, e.g., Panayotou et al, Mol. Cell. Biol., 13: 3567-3576 (1993)] or radioactive ligand based assays. The former has a relatively low throughput, while the latter requires cumbersome filtration manipulations and generates radioactive waste, an increasingly difficult disposal issue.
  • Biacore surface plasmon resonance
  • the former has a relatively low throughput, while the latter requires cumbersome filtration manipulations and generates radioactive waste, an increasingly difficult disposal issue.
  • the present invention addresses this need by providing novel materials and methods for in vitro competitive binding assays for identifying substances which inhibit or interfere with the binding together of pairs of proteins capable of mutual association, i.e., binding, to form binding complexes.
  • assays for identifying compounds capable of inhibiting the binding of intracellular proteins or protein domains especially those involved in cellular signal transduction with their binding partners.
  • proteins include, for instance, proteins which contain one or more SH2 domains, PI domains, SH3 domains, or WW domains, each with its respective protein ligand.
  • the invention provides an in vitro assay method for identifying a test substance which inhibits the mutual association of a first protein to a second protein.
  • the method includes the steps of preparing a mixture containing the first protein, the second protein bearing a covalently linked fluorophore, and at least one test substance.
  • the mixture is irradiated with polarized light of a suitable wavelength permitting excitation of the fluorophore as indicated by emission of polarized light.
  • the degree of polarization of the emission is measured and the effect of the presence or concentration of the test substance is determined.
  • Inhibitory activity of the test substance is shown by a decrease in the observed emission polarization values of the mixture of the first and second proteins in the presence of the text substance as compared with the same protein mixture in the absence of the test substance.
  • Inhibition of protein:protein association can result from binding a test substance to the first protein or to the labeled ligand protein or peptide.
  • the assay method can be viewed as a method for identifying a test substance which competitively binds to either member of the binding pair.
  • the degree of polarization of the emission is measured, and the effect of the presence or concentration of the test substance in decreasing the observed emission polarization is observed and compared with a mixture in the absence of the test substance.
  • Competitive binding of the test substance correlates with decreased depolarization values.
  • the invention provides an inhibitor of the association of a first protein with a second protein, first identified by the methods above.
  • the invention provides components or reagents, e.g., a protein bearing a covalently linked fluorophore, useful in the methods of the invention.
  • the components or reagents can further be packaged in a kit with instructions for use in the described methods.
  • Fig. 1 depicts the structure of a fluorescent probe FMT1 , described in Example 2.
  • Fig. 2A is the saturation curve of FMT1 binding to Src SH2, which plots bound Src/total tracer vs. total Src concentration.
  • Calculated Kd is 0.24 with an error of 0.008 ⁇ M, (Chi) 2 of 0.0027, R of 0.9987.
  • Fig. 2B is the Scatchard analysis (transformation) of the data in Fig. 2 A, plotting Rb/Rf vs. Bound.
  • Calculated Kd is 0.26 ⁇ M, with an R value of 0.992 for the linear fit.
  • Fig. 3 is an Src competition curve (2% DMSO) plotting % probe binding vs. inhibitor concentration for the following tetrapeptides: Ac-pYEEI (open circle); Ac- pYpYEEI (closed square); Ac-pYGGL (plus sign); Ac-pYEDL (open triangle); Ac- DGVpYTGL (closed triangle).
  • Fig. 4A is a depiction of a 96 well plate of the experiment of Example 5, long format, with an arrow illustrating the direction of dilution. Clear circles are sample wells, gray circles are wells containing probe alone and dark circles are wells containing probe and protein.
  • Fig. 4B is a depiction of a 96 well plate of the experiment of Example 5, short format, with an arrow illustrating the direction of dilution per 4 rows of wells. Circles are defined as in Fig. 4A.
  • Fig. 5 depicts the structure of an alternative fluorescent probe for use in the Src- SH2 assay, described in Example 2.
  • Fig. 6A is a depiction of performance of the FP assay with no inhibitor present.
  • the fluorescein-labeled second protein (or probe) binds to its binding partner (first protein having an SH2 domain). Light from the vertical polarized light source remains polarized due to the slow rotation of the bound complex.
  • Fig. 6B is a depiction of performance of the FP assay with inhibitor present.
  • the inhibitor binds to the SH2 domain-containing first protein, thereby preventing binding of the fluorescein-labeled second protein (or probe).
  • the small unbound probe rotates more quickly than does the complex of Fig. 6A. Light from the vertical polarized light source becomes depolarized due to the quick rotation of the unbound probe.
  • the present invention addresses the needs of the art by providing a fluorescence polarization (FP)-based assay, for identifying and measuring the capacity of a test substance to disrupt or inhibit the association between a pair of proteins.
  • FP fluorescence polarization
  • the assay of the present invention is designed to enable one to detect an inhibitor of any proteimprotein interaction.
  • proteimprotein interaction or “mutual association” of proteins is meant any complex or binding, covalent or non-covalent, which naturally forms between two different proteins.
  • protein:protein association involves the complex formed between a receptor and its naturally-occurring ligand. Interactions between fragments of proteins, i.e., peptides, with another protein or peptide are also encompassed by the term proteimprotein interaction.
  • proteimprotein binding abound in the art, e.g., the binding between an antibody and a protein antigen or epitope, the binding between a cell-surface receptor and its protein ligand, the binding of various signalling proteins with their protein binding pairs, etc.
  • proteimprotein interaction which are used herein to demonstrate the method of this invention involve, as a first protein, a protein containing one or more SH2 domains, and/or SH3 domains (Syk, Zap, Src and Lek) and, as a second protein, a ligand for that first protein.
  • a protein containing one or more SH2 domains, and/or SH3 domains Syk, Zap, Src and Lek
  • a ligand for that first protein for additional background information on Zap and Syk proteins and their SH2 domains, and peptide ligands, see, International Patent Application Nos. PCT/US96/13918, inco ⁇ orated herein by reference.
  • the pu ⁇ oses of the present invention and for using currently conventional detection equipment, it is preferred to use a first protein/peptide that has a significantly greater molecular weight than the second protein or peptide, which naturally interacts with it.
  • the larger protein which participates in the protein:protein interaction is referred to as the first protein.
  • the second protein or peptide which is labelled with the fluorophore according to the assay method.
  • the first protein of the binding pair has a molecular weight of at least 2 to about 100 times greater than the molecular weight of the labeled second protein/peptide of the binding pair.
  • the first protein of the binding pair has a molecular weight of at least 25 to about 50 times greater than the molecular weight of the labeled second protein/peptide of the binding pair.
  • the first protein need not necessarily be rigorously purified in production, as described below, but it is important to know the concentration of the first protein for certain quantitative pu ⁇ oses, e.g., for the construction of a saturation curve or to determine Kd values for the affinity of the two protein components.
  • One specifically exemplified "first protein" of the examples contains an SH2 domain which serves as a receptor for a tyrosine phosphorylated peptide.
  • a receptor may be a protein, a fusion protein, polypeptide, peptide or fragment thereof which contains a "phosphopeptide binding domain" (PBD).
  • a PBD is a receptor domain present, e.g., in certain signaling proteins, which is capable of binding to a phosphorylated protein or phosphopeptide and thereby of directing protein-protein or protein-peptide association.
  • an "SH2 domain" is one such receptor domain.
  • the receptor can be a polypeptide containing one or more SH2, or other phosphopeptide binding domains.
  • the receptor may be located within a larger protein, or may be a peptide fragment thereof.
  • the receptor is preferably of human or other animal origin.
  • first protein contains an SH3 domain which serves as a receptor for a corresponding peptide sequence or motif.
  • a receptor may be a protein, a fusion protein, polypeptide, peptide or fragment thereof which contains an SH3 or SH3-like domain.
  • first protein contains both an SH2 and an SH3 domain.
  • the second protein is a ligand (naturally-occurring or otherwise) of the first protein or receptor.
  • the second protein is generally the smaller of the two proteins in the binding pair and is preferably the protein of the pair which is labeled with a fluorescent moiety, thereby forming the "probe" component useful in the methods described herein.
  • the second protein or "ligand” is a protein, fusion protein, peptide or fragment thereof which contains one or more tyrosine residues, which is capable of binding selectively and with specificity to a phosphopeptide binding domain when at least one of the peptide ligand's tyrosine residues is phosphorylated.
  • An "SH2 ligand” is an example of such a ligand.
  • the probe peptide or protein binds to its (usually) larger binding partner with a Kd in the range of about 0.1 to about 1000 nM. More desirably, the two binding partners bind to each other with a Kd better than (i.e., numerically smaller than) about 300 nM, more preferably with a Kd in the range of about 5 to about 50 nM.
  • DNA sequence information and expression technology is available which permits recombinant production of any desired protein(s)/peptide(s) using a variety of expression systems.
  • To produce the proteins used in these assays one may express DNAs encoding the whole protein or a portion of the protein containing at least a domain of interest.
  • the protein or portion of the protein may be expressed as a fusion protein, also by conventional techniques, especially in the case of the larger of the two binding proteins.
  • any materials and methods conventional for producing a protein may be used including both prokaryotic and eukaryotic systems.
  • proteins/peptides may be expressed by baculovirus, bacterial, yeast or mammalian expression systems, whether as full-length proteins, fragments containing the receptor domain(s) or as fusion proteins.
  • Such expression systems are conventional in the art. See, for examples, the descriptions in Sambrook et al, Molecular Cloning. A Laboratory Manual., 2d edit., Cold Spring Harbor Laboratory, NY (1989).
  • expression vectors for a protein or domain of interest can be constructed by ligating into a conventional expression vector the DNA sequence encoding the desired protein, protein domain or, if known, a consensus homology domain for the domain of interest, alone or preferably with additional flanking sequence.
  • additional flanking amino acids enhance stability, improve expression levels, improve its ability to interact with ligands or other proteins or be necessary or desirable for linking to a fusion protein for reasons discussed below.
  • the SH3 consensus homology domain includes amino acids 91-140.
  • the desired protein or protein domain may be expressed within all or part of its natural context, as an isolated domain, in a tandem array containing two or more of the same or different domains, or as a fusion protein with other unrelated domains including but not limited to SH2-like domains, protein kinase domains, glutathinone S-transferase (GST), epitope tags, kinase recognition sequences, maltose binding protein, signal sequences, biotin-modification sequences, etc.
  • SH2-like domains protein kinase domains
  • GST glutathinone S-transferase
  • epitope tags kinase recognition sequences
  • maltose binding protein signal sequences
  • biotin-modification sequences etc.
  • the proteins or protein domains may be modified to: facilitate purification e.g. by expression as a fusion to glutathione-S-transferase, maltose binding protein, metal-chelation sequences (poly-histidine), protein A or others; facilitate identification or quantitation, e.g. by covalent modification using biotin, fluorophores, chromophores, scintillons, spin labels, radioactive or non-radioactive isotope tags, magnetic particles, metal coloids, etc; adhere to defined solid supports, e.g. by expression as a fusion to an epitope tag or other antigenic domain; engineered to provide unique or uniquely accessible protein features e.g.
  • N-terminal serine, cysteine, lysine or others, etc. remove undesirable features that pose experimental complications, e.g. by mutation of cysteines that participate in unnatural domain dimerization improve stability under conditions of binding assays (e.g. by altering the natural coding sequence to encode cysteines that form stabilizing disulfides).
  • test substance or inhibitor is defined herein as a compound or composition which binds selectively to either the first protein or the second protein which participate in the proteimprotein interaction.
  • the test substance selectively blocks or otherwise inhibits the interaction between these two proteins.
  • this inhibitor can bind to the first protein with competitive avidity vis-a-vis its naturally occurring binding protein, or it can bind the second protein.
  • the two proteins are exemplified as a tyrosine phosphorylated receptor and its naturally occurring ligand
  • the inhibitor can bind to the receptor competitively with the ligand, or it can selectively block or otherwise inhibit the interaction between the receptor and ligand normally mediated by one or more tyrosine phosphorylated peptides or domains.
  • Test substances or compositions to be assessed for their ability to bind selectively to the first or second protein of interest can be obtained from a variety of sources, including, for example, microbial broths, cellular extracts, conditioned media from cells, synthetic compounds and combinatorial libraries.
  • the assay method of this invention may be used to screen natural product and test compound libraries or structurally-biased diversity libraries to identify desired inhibitors.
  • the test substance may be selected from a mixture of one or more test peptides, wherein said mixture is provided in the form of a library of synthetic peptides or in the form of a phage library displaying the various peptides.
  • a “fluorophore” or “fluorescent moiety” is a fluorescent molecule which, in solution and upon excitation with polarized light, emits light back into a fixed plane (i.e., the light remains polarized). Numerous known fluorescent labeling moieties of a wide variety of structures and characteristics are suitable for use in the practice of this invention. Similarly, methods and materials are known for covalently linking them to other molecules [see, e.g., Richard P. Haugland, Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals 1992-1994 (5th edit, 1994, Molecular Probes, Inc.)].
  • fluorophore In choosing a fluorophore, it is preferred that the lifetime of the fluorophore's exited state be long enough, relative to the rate of motion of the labeled probe or peptide, to permit measurable loss of polarization following emission.
  • Suitable fluorophores include fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, and umbelliferone.
  • a fluorophore having an excitation wavelength and emission wavelength in the visible rather than ultraviolet range of the spectrum is typically preferred to use a fluorophore having an excitation wavelength and emission wavelength in the visible rather than ultraviolet range of the spectrum to avoid possible interference from test compound fluorescence.
  • the fluorophore is covalently linked to the smaller protein, i.e., the second protein, to be labeled, e.g., a peptide ligand, using a sufficiently short linker to avoid introducing undue motion to the fluorophore, i.e., motion not correlated to the motion of the labelled peptide.
  • Example 2 provides a description of the method used by these inventors in which a fluorescent moiety is chemically attached by covalent bonds onto a second protein molecule (a peptide ligand).
  • a fluorescent moiety is chemically attached by covalent bonds onto a second protein molecule (a peptide ligand).
  • a peptide ligand a second protein molecule
  • FP first described by Perrin, J. Phys. Rad.. 1:390-401 (1926), is based upon the finding that the emission of light by a fluorophore can be depolarized by a number of factors, the most predominant being rotational diffusion, or, in other words, the rate at which a molecule tumbles in solution.
  • “Polarization” is the measurement of the average angular displacement of the fluorophore which occurs between the abso ⁇ tion and subsequent emission of a photon. This angular displacement of the fluorophore is, in turn, dependent upon the rate and extent of rotational diffusion during the lifetime of the excited state, which is influenced by the viscosity of the solution and the size and shape of the diffusing fluorescent species.
  • the polarization is directly related to the molecular volume or size of the fluorophore.
  • the polarization value is a dimensionless number (being a ratio of vertical and horizontal fluorescent intensities) and is not affected by the intensity of the fluorophore.
  • SH2 domain refers to a sequence which is substantially homologous to a Src homology region 2 (SH2 region).
  • Src homology region 2 is a noncatalytic domain of -100 amino acids which was originally identified in the viral Fps and viral Src cytoplasmic tyrosine kinases by virtue of its effects on both catalytic activity and substrate phosphorylation (T. Pawson, Oncogene 3, 491 (1988) and I. Sadowski et al., Mol. Cell. Biol. 6, 4396 (1986)).
  • SH2 domains have been found in a variety of eukaryotic proteins, some of which function in intracellular signal transduction. Many arc known in the art.
  • SH2 domain-containing proteins examples include (1) members of the src-family protein tyrosine kinases (Src, Lyn, Fyn, Lek, Hck, Fgr, Yes), (2) She (3) Tsk, (4) Btk, (5) VAV, (6) Grb2, (7) Crk. and (8) signal transducer and transcription (STAT) proteins.
  • a number of proteins such as ZAP-70, p85 phosphatidylinositol 3' kinase (PI3K), Syk, GTPase Activating Protein (GAP), and Phospholipase C gamma, have two SH2 domains.
  • SH2 domain-containing proteins have been identified in human, rodent, sheep, bovine, C. elegans, Drosophila, Xenopus, flatworm, freshwater sponge, and hydra.
  • RNA or protein sequence is by using one of many available computer alignment programs.
  • One example is pfscan, which can be run via the World Wide Web (WWW) site at http://ulrec3.unil.ch/software/profilescan.html.
  • WWW World Wide Web
  • a protein sequence is tested against a "profile" describing the SH2 domain motif.
  • the particular strength of profiles is that they can be used to describe very divergent protein motifs.
  • a profile identifies which types of residues are allowed at what position within the domain, which amino acids are conserved, which ones are not, which positions or regions can allow insertions, and which regions may be dispensable. Additional information on Pfscan and PROSITE can be obtained at the web page http://ulrec3.unil.ch/index.html operated by the Bioinformatics Group at the ISREC (Swiss Institute for Experimental Cancer Research). As an example we analyzed the peptide sequence of human Src with the pfscan program. The results are shown below. The program clearly identified the SH2 domain of Src as encompassing the region from amino acids 150-247 of the Src peptide sequence. In addition, the SH3 and kinase domains were identified by pfscan.
  • the NScore of a match is the negative decadic logarithm of the expected number of matches of the given quality (or better) in a random database of the given size. For NScores «1 this converges to the probability of finding the match in the database. Since the number of expected matches depends on the size of the database, the decadic logarithm of the database size must be subtracted before the calculation:
  • the following table gives somes examples on how to convert the NScores into probabilities for the SwissProt database and the nonredundant (nr) protein database. The calculation is based on a database size of
  • the segment of a test sequence contains an SH2 domain with an SH2 profile NScore value > 7.5, preferably > 8, more preferably > 9, more preferably > 10.
  • the N-terminal 160 amino acid sequence from human ZAP-70 was applied to pfscan.
  • SH2 domains can be identified using other computer alignment programs, such as MegAlign within the DNAstar computer package (Madison, WI). To do this, one or more known SH2 domains and a test sequence are aligned by the clustal method. A sequence having 3 25%, in some cases 30 - 50 %, in other cases > 50%, amino acids identical to a known SH2 domain is identified as an SH2 homology domain. The positions of identical amino acids between the test sequence and different known SH2 domains can vary, except for one position. All SH2 domains identified to date have a conserved arginine residue approximately 25-40 residues from the start of the SH2 homology domain. In human src this arginine is found within the sequence FLVRES, where abbreviations for the amino acid residues are: F, Phe; L, Leu; V, Val; R, Arg; E, Glu; S, Ser.
  • SWISS-PROT database can be accessed over the WWW at EBI http://www.ebi.ac.uk. For example, in the file listed for human Src (P12931 ), the region containing the SH2 domain is shown to be 150-247.
  • SH2 or SP I2-like domains may be accomplished by screening a cDNA expression library with a phosphorylated peptide ligand for a known SH2 domain to isolate cDNAs for SH2 proteins.
  • the SH2 domain or protein containing the SH2 domain may be isolated from naturally occuring sources (e.g. cells, tissues, organs, etc); produced recombinantly in bacteria, yeast or eukaryotic cells; produced in vitro using cell free translation systems; or produced synthetically (e.g. peptide synthesis).
  • SH2 or SH2-like domains may not be identified via the pfscan program nor exhibit significant homology with known SH2 domain sequences to be detected by computer alignment programs. These sequences may, nevertheless, exhibit the same or similar three-dimensional structure as known SH2 domains and function as an SII2-like domain and function to bind phosphotyrosine-containing peptides or proteins.
  • the three- dimensional structure of several known SH2 domains have been determined.
  • SH2 domains are characterized as two anti-parallel beta sheets composed of 5 or 6 beta strands. Regions forming an alpha helix may or may not be present within the domain.
  • SH2 or SH2-like domains may be recognized as having an SH2-like domain structure when solved by x-ray crystallography or NMR spectroscopy. Alternatively, a predicted structure by homology modeling may be used to identify a particular protein sequence as an SH2-like domain.
  • the alignment of SH2 domains used to generate the SH2 profile for pfscan is based on alignment of approximately 390 SH2 domains from proteins of various species.
  • the list of proteins containing SH2 domains used in the alignments in the Swiss-Prot Database includes the following (P##### is the Swiss-Prot Database Accession number):
  • P29350 PTN6_HUMAN P29351, PTN6_MOUSE P29353, SHC_HUMAN
  • SH2 and SH2-like domains as described in the foregoing paragraphs may be used in the practice of this invention. Using information provided herein, and by analogy to the examples provided below, one may carry out this invention with any SH2 domain, SH2-like domain, PID or PID-like domain and a peptide ligand therefor, e.g. in place of ZAP, Syk, Src or Fyn SH2 domains.
  • PID phosphotyrosine interaction domain
  • the coding sequence for a PID domain is substituted in the appropriate vector for the SH2 domain coding sequence and a ligand that recognizes the PID domain replaces the SH2 domain ligand.
  • Phosphorylation of the PID ligand could be accomplished using v-Src, as described herein.
  • Alternative protein kinases could be used to phosphorylate the PID ligand.
  • a protein kinase endogenous within the cell could catalyze phosphorylation of the PID ligand.
  • the phosphotyrosine interaction domain (PI domain or PID)[3] is the second phosphotyrosine-binding domain found in the transforming protein She [1 ,2].
  • She couples activated growth factor receptors to a signalling pathway that regulates the proliferation of mammalian cells and it might participate in the transforming activity of oncogenic tyrosine kinases.
  • the PI domain specifically binds to the Asn-Pro-Xaa- Tyr(p) motif found in many tyrosine-phosphorylated proteins including growth factor receptors.
  • PID has also been found in the She related protein Sck [1 ] and several otherwise unrelated regulatory proteins [3] which are listed below.
  • Mammalian She (46 kD and 52 kD isoforms) contains one N-terminal PID, a collagen-like domain and a C-terminal SH2 domain.
  • Human She related protein Sck contains one PI domain and a SH2 domain.
  • Mammalian XI 1 is expressed prominently in the nervous system. It contains 2 disc homologous regions (DHR) of about 100 AA downstream of the PID.
  • DHR disc homologous regions
  • Drosophila nuclear Numb protein is required in determination of cell fate during sensory organ formation in drosophila embryos. It has one PID.
  • Caenorhabditis hypothetical protein F56D2.1 contains an N-terminal metal loproteinase domain followed by one PID.
  • Rat FE65 The WW domain as well as the 2 PIDs found in the sequence of FE65 indicate that this protein is probably involved in signal transduction.
  • Drosophila protein disabled is a cytoplasmic, tyrosine phosphorylated protein found in CNS axons and body wall muscles. It is involved in embryonic neural development. It contains one N-terminal PI domain.
  • Human EST05045 protein fragment has one PID.
  • a PI domain alignment based on a number of PI domains from various species is illustrated in the WWW site at http://ulrec3.unil.ch/prf_details/alignments/PID.msf. Another such alignment is shown at the web site at http://www.bork.embl- heidelberg.de/Modules/pi-ali.html.
  • SH3-like domain refers to a sequence which is substantially homologous to a Src homology region 3 (SH3 region).
  • Src homology 3 region is a noncatalytic domain of -60 amino acids which was originally identified in the viral Fps and viral Src cytoplasmic tyrosine kinases by virtue of its effects on both catalytic activity and substrate phosphorylation (T. Pawson, Oncogene 3, 491 (1988) and I. Sadowski et al., Mol. Cell. Biol. 6, 4396 (1986)).
  • SH3 domains have been found in a variety of eukaryotic proteins, some of which function in intracellular signal transduction.
  • SH3 domain-containing proteins examples include (1) members of the src-family protein tyrosine kinases (Src, Lyn, Fyn, Lek, Hck, Fgr, Yes), (2) Grb-2, which has two SH3 domains, (3) Sprk, a threonine/serine protein kinase, (4) Tsk, (5) Btk, (6) Vav, (7) GTPase Activating Protein (GAP), (8) p40, p47, and p67 proteins of the neutrophil oxidase complex, and (9) phosphatidylinositol 3' kinase, (10) Crk, (1 1) phospholipase C gamma, (12) Abl.
  • Src src-family protein tyrosine kinases
  • Grb-2 which has two SH3 domains
  • Sprk a threonine/serine protein kinase
  • Tsk (5)
  • SH3 domain-containing proteins have been identified in human, rodent, bovine, C. elegans, and yeast. Other SH3 domains may be selected from the scientific literature or identified by sequence analysis or cloning by the methods described above. See e.g. PCT/US95/03208 for a wealth of background information relating to SH3 domains and their ligands.
  • SH3 or SH3-like domains may not match any of the 18 conserved amino acids nor exhibit significant homology with known SH3 domain sequences to be detected by computer alignment programs. These sequences may, nevertheless, exhibit the same or similar three-dimensional structure as known SH3 domains and function as an SH3-like domain.
  • the three-dimensional structure of several known SH3 domains have been determined.
  • SH3 domains are characterized as two anti-parallel beta sheets composed of 5 or 6 beta strands. Regions forming an alpha helix may or may not be present within the domain.
  • SH3 or SH3-like domains may be recognized as having an SH3-like domain structure when solved by x-ray crystallography or NMR spectroscopy. Alternatively, a predicted structure by homology modeling may be used to identify a particular protein sequence as an SH3-like domain.
  • the in vitro assay method of this invention utilizes FP for identifying a test substance which competitively binds to, or inhibits the mutual association of, a first protein molecule to a second protein molecule.
  • Fluorescence polarization is an extremely useful method for studying ligand-protein and protein-protein interaction.
  • the present invention is based upon the observation that changes in polarization will occur if a fluorescent molecule undergoes a molecular weight change due to cleavage or binding to another molecule.
  • This intrinsic property of the fluorescent moiety is utilized in the assay of this invention. According to this method, a mixture is made which contains the following components:
  • a selected amount of a first protein molecule e.g., a tyrosine phosphorylated receptor, which is capable of binding or otherwise mutually associating with a smaller second protein molecule;
  • This mixture is accomplished under conditions suitable to permit complex formation between the first and second proteins, if they were admixed in the absence of test substance.
  • the conditions are also suitable to permit its competitive binding to the first or second proteins.
  • This mixture of (a), (b) and (c) is irradiated with plane polarized light of a wavelength which is sufficient to excite the fluorophore.
  • the light subsequently emitted by the fluorescent second protein is polarized to varying degrees depending on the molecular volume of the fluorescent second protein. In the unbound state in solution, low molecular weight peptides rotate rapidly, and give low polarization readings.
  • the lower-molecular weight fluorescent second protein When in the presence of its binding/interacting first protein partner, e.g., a receptor, the lower-molecular weight fluorescent second protein binds to the higher molecular weight first protein, e.g., a tyrosine phosphorylated protein or peptide receptor.
  • first protein e.g., a tyrosine phosphorylated protein or peptide receptor.
  • the labeled second protein binds to its target first protein and is illuminated by plane polarized light, the large first proteimsecond protein complex tumbles more slowly, and the polarization readings increase.
  • the method of this invention thus follows changes in the ratio of polarization in the horizontal and vertical planes of the emission wavelength range. This is in distinct contrast to following changes in the intensity of absorbance within a particular wavelength range, which is the way conventional fluorescent labels are used.
  • the change measured by the present invention is a direct measure of the binding of the labeled second protein to the first protein.
  • This difference in polarization values of free labeled second protein vs. bound second proteimfirst protein complex is used to measure the bound and free ratios of the second protein and analyze its binding to the first protein when in the presence of a test substance. Such measurement may occur in either saturation or competition experiments.
  • the FP assay of this invention can thus be used in many solutions, including in the cytoplasm of the cell.
  • the degree of polarization of the emission is measured without the necessity to separate the components in the mixture. Finally, the effect of the presence or concentration of the test substance is determined by comparing the ratio of the polarization levels of the mixture with the polarization levels of the same amounts of the first and second proteins/peptides in the absence of test compound.
  • the second protein will remain free in solution and low polarization will be measured. If the test substance is not an inhibitor or a good inhibitor, the complex will be formed and the polarization of the mixture will increase. Thus a decrease in the observed emission polarization depolarization values from known polarization levels of the first protein :second protein complex in the absence of test compound is noted in the presence of an inhibitor test substance.
  • the method of this invention follows changes in the ratio of polarization in the horizontal and vertical planes of the emission wavelength range, rather than changes in the intensity of absorbance within a particular wavelength range, the method is less vulnerable to interference from high absorbance of test compounds in solution.
  • the methods of this invention are susceptible to automation. For example, all or several of the steps outlined above may be performed by an apparatus programmed to conduct automatically two or more steps for a given test substance or one or more steps for a plurality of test substances or test substance concentrations.
  • any standard fluorometer equipped for polarization experiments or measurements may be used in practicing this invention to both irradiate the mixture and measure the polarization.
  • Wavelengths suitable to excite the fluorophore depend on the nature of the fluorophore, as described above. Typically, one uses cut off filters to define a wavelength range which is determined by the excitation and emission wavelengths of the fluorophore.
  • fluorescein carboxyamide peptides For fluorescein carboxyamide peptides, one would typically use an excitation cutoff filter of 485nM. Also, non-polarizing material should be used for any component of the apparatus, including the test chambers in which samples are evaluated, which will be in the light path. Plastics and fiber optics are generally avoided in such uses in favor of optical glasses, quartz, etc.
  • Such fluorometers have been optimized for polarization measurements and have much higher sensitivity than standard fluorometers.
  • Other automated equipment may provide both the admixing step combined with the other steps, and/or the comparison of the polarization of the control mixture without the test substance and the test mixture with the test substance.
  • One of skill in the area of automation may use various apparatus to substantially automate the assays of this invention.
  • the invention provides components or reagents, e.g., a protein bearing a covalently linked fluorophore, useful in the methods of the invention.
  • the components or reagents can further be packaged in a kit with instructions for use in the described methods.
  • a compound Once a compound has been identified as an inhibitor, it can be produced using known methods, such as by recombinant methods of protein production or chemical synthesis. It can also be obtained from the source in which it was initially identified.
  • test compounds identified as inhibitors by the method of this invention may be further evaluated for binding activity with respect to one or more additional proteins of interest, or with respect to additional proteins containing the domain(s), using various approaches, a number of which are well known in the art.
  • the counterscreen may be carried out using the methods and materials of the subject invention, or may be conducted using alternative approaches for the detection of direct or competitive binding, including, e.g., cell-based assays or surface plasmon resonance (BIAcore®) technology [see, e.g., Panayotou et al, Mol. Cell. Biol.. _3: 3567-3576 (1993)].
  • BIOS surface plasmon resonance
  • test compounds identified in the assay system of this invention can be further evaluated by conventional methods for assessing toxicological and pharmacological activity.
  • test compounds identified as inhibitors may further be evaluated for activity in inhibiting cellular or other biological events mediated by a pathway involving the proteimligand interaction of interest using a suitable cell-based assay or an animal model.
  • Cell-based assays and animal models suitable for evaluating inhibitory activity of a test compound with respect to a wide variety of cellular and other biological events are known in the art. New assays and models are regularly developed and reported in the scientific literature.
  • compounds which bind to an SH2 domain involved in the transduction of a signal leading to asthma or allergic episodes may be evaluated in a mast cell or basophil degranulation assay.
  • the inhibitory activity of a test compound identified as an SH2 inhibitor by the method of this invention with respect to cellular release of specific mediators such as histamine, leukotrienes, hormonal mediators and/or cytokines, as well as its biological activity with respect to the levels of phosphatidylinositol hydrolysis or tyrosine phosphorylation can be characterized with conventional in vitro assays as an indication of biological activity.
  • mediators such as histamine, leukotrienes, hormonal mediators and/or cytokines
  • histamine release can be measured by a radioimmunoassay using a kit available from AMAC Inc. (Westbrook, ME).
  • AMAC Inc. Westbrook, ME
  • inhibitors identified by this invention may also be tested in an ex vivo assay for their ability to block antigen-stimulated contraction of sensitized guinea pig tracheal strip tissue. Activity in this assay has been shown to be useful in predicting the efficacy of potential anti-asthma drugs.
  • inhibitors identified by the method of this invention which bind to a protein involved in the transduction of a signal involved in the initiation, maintenance or spread of cancerous growth may be evaluated in relevant conventional in vitro and in vivo assays. See e.g., Ishii et al., J. Antibiot., XLIL1877-1878 (1989); and US Patent 5,206,249 (issued 27 April 1993).
  • Inhibitors identified by this invention may be used as biological reagents in assays as described herein for functional classification of a particular protein, particularly a newly discovered protein. Families or classes of proteins may thus be defined functionally, with respect to ligand specificity. Moreover, inhibitors identified by this invention can be used to inhibit the occurrence of biological events resulting from molecular interactions mediated by a the protein or protein: ligand pair of interest. Inhibiting such interactions can be useful in research aimed at better understanding the regulation and biological significance of such events.
  • inhibitory agents would be useful, for example, in the diagnosis, prevention or treatment of conditions or diseases resulting from a cellular process(es) mediated by a targeted interaction.
  • a patient can be treated to prevent the occurrence or progression of osteoporosis or to reverse its course by administering to the patient in need thereof an SH2 binding or blocking agent which selectively binds Src SH2.
  • phosphopeptide binding or blocking agents may be useful therapeutically, including, e.g., breast cancer where the SH2 domain-containing proteins Src, PLCgamma and Grb7 have been implicated.
  • Other relevant conditions include prostate cancer, in which case targeting Grb2, PLCg, and PI3K, all of which contain SH2 domains, may be useful in treatment or prevention of the disease.
  • Inhibition of the interaction of Grb2 or Abl SH2 domains with Bcr-abl may be useful to treat chronic myelogenous leukemia (CML) or acute myelogenous leukemia (AML).
  • CML chronic myelogenous leukemia
  • AML acute myelogenous leukemia
  • an PBP inhibitor would be to prevent interferon-, growth factor-, or cytokine-mediated diseases (e.g. inflammatory diseases) by targeting the PBDs of STAT proteins.
  • agents that block the SH2 domains of ZAP-70, which is involved in activation of T-cells, would be useful in the treatment of autoimmune diseases.
  • An inhibitor that blocks one or both SH2 domains of ZAP-70 would also be useful as an immunosuppressant to prevent rejection of skin and organ transplants.
  • SH3 inhibtors would be useful in the diagnosis, prevention or treatment of conditions or diseases resulting from a cellular processes mediated by an SH3-based interaction.
  • a patient can be treated to prevent the occurence or progression of osteoporosis or to reverse its course by administering to the patient in need thereof an SH3 inhibitor which selectively binds to or inhibits interactions with src SH3.
  • SH3 inhibitors can be used therapeutically, including restenosis, rheumatoid arthritis, gout, asthma, emphysema, immune vasculitis, ulcerative colitis, psoriasis and acute respiratory distress syndrome, in which an SH3 of neutrophil oxidase p47 and p67 complex has been implicated.
  • Other relevant conditions include chronic myelogenous leukemia, in which case SH3 domains of Grb-2 are targeted. It has recently been shown that the BCR-abl oncogene in CML participates in the ras pathway for growth stimulation through its interaction with Grb-2.
  • Grb-2 SH3 domains In these cells, inhibition of the interaction of Grb-2 SH3 domains with the SOS oncogene will block its ability to stimulate cell proliferation. Still other relevant conditions include cancers such as breast cancer, glioblastomas, head and neck tumors and ovarian tumors, for which the SH3 domain of Grb-2 would be targeted. For example, tumors with associated amplification of receptors for EGF and PDGF could be inhibited by blocking activation of the Ras pathway through inhibition of the interaction between Grb-2 (SH3)and Ras.
  • SH3 Grb-2
  • an SH3 inhibitor identified by the subject invention could be administered to a patient in need thereof to suppress immune function.
  • An inhibitor of a proteindigand interaction identified by the method of this invention can be formulated into a pharmaceutical composition containing a pharmaceutically acceptable carrier and/or other excipient(s) using conventional materials and means.
  • a composition can be administered to an animal, either human or non-human, for therapy of a disease or condition resulting from cellular events involving the targeted protein-ligand interaction. Administration of such composition may be by any conventional route (parenteral, oral, inhalation, and the like) using appropriate formulations as are well known in this art.
  • the inhibitor can be employed in admixture with conventional excipients, ie, pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral administration.
  • compositions Inhibitors identified by this invention can be formulated into pharmaceutical compositions containing a therapeutically (or prophylactically) effective amount of the inhibitor in admixture with a pharmaceutically acceptable carrier and/or other excipients (i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral administration) using conventional materials and means.
  • a pharmaceutically acceptable carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the carrier and composition can be sterile. The formulation should suit the mode of administration.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ease pain at the side of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • Topical compositions include a pharmacologically acceptable topical carrier, such as a gel, an ointment, a lotion, or a cream, which includes, without limitation, such carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils.
  • topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water.
  • Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.
  • the invention provides methods of treating, preventing and/or alleviating the symptoms and/or severity of a disease or disorder referred to above by administration to a subject of the inhibitor in an amount effective therefor.
  • the subject will be an animal, including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human.
  • mammals is meant rodents such as mice, rats and guinea pigs as well as dogs, cats, horses, cattle, sheep, nonhuman primates and humans.
  • Such effective amounts can be readily determined by evaluating the inhibitors identified by this invention in conventional assays well-known in the art, including assays described herein.
  • compositions may be by any conventional route using appropriate formulations as are well known in this art.
  • Various delivery systems are known and can be used to administer the inhibitor, e.g., encapsulation in liposomes, microparticles, microcapsules.
  • One mode of delivery of interest is via pulmonary administration.
  • Other methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, nasal and oral routes.
  • the inhibitor may be administered by infusion or bolus injection, by abso ⁇ tion through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
  • Administration can be systemic or local.
  • preferred routes of administration are oral, nasal or via a bronchial aerosol or nebulizer.
  • Administration to an individual of an effective amount of the inhibitor can also be accomplished topically by administering the compound(s) directly to the affected area of the skin of the individual.
  • the inhibitor may be disposed within devices placed upon, in, or under the skin. Such devices include patches, implants, and injections which release the compound into the skin, by either passive or active release mechanisms.
  • the amount of the inhibitor which will be effective in the treatment or prevention of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • a typical effective dose of the inhibitor is in the range of about 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10 mg/kg of mammalian body weight, administered in single or multiple doses.
  • the inhibitor may be administered to patients in need of such treatment in a daily dose range of about 1 to about 2000 mg per patient.
  • the precise dosage level of the inhibitor, as the active component(s), should be determined by the attending physician or other health care provider and will depend upon well known factors, including the phosphopeptide binding interaction under consideration, the route of administration, and the age, body weight, sex and general health of the individual; the nature, severity and clinical stage of the disease; and the use (or not) of concomitant therapies.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • physiologically acceptable surfactants e.g., glycerides
  • excipients e.g., lactose
  • carriers e.g., diluents
  • amino acid couplings four equivalents of amino acid, four equivalents of coupling reagent, 2-(lH-benzotriazole-l - yl)-l,l,3,3-tetramethyluronium tetrafluoroborate (TBTU), and eight equivalents of N- methylmo ⁇ hoiine (NMM) were used per equivalent of amine on the resin.
  • Amino acids used were Fmoc-Gly, Fmoc-Glu(Tbu), Fmoc-IIe, Fmoc-Thr(Tbu), and Fmoc-Tyr(Tbu). Fmoc deprotection was done using 20% piperidine in DMA.
  • Peptides were phosphorylated on the solid-phase using standard methodology fsee, e.g.. Merrifield. J. Am. Chem. Soc. 85:2149-2154 (1963)1. Resins and 1-l .H- tetrazole were dried under vacuum over P 2 O 5 overnight. To a portion of resin (0.1-0.5 g) mixed with 50 equivalents of l-l,H-tetrazole was added 1 ml/O.lg of dry DMA. The resin was stirred and swelled for 20 minutes.
  • peptides were purified in the following manner. Crude lyophilized peptides were dissolved in DMSO at a concentration of 100-300 mgs per ml. Peptides were purified on a Semi-Preparative reverse phase HPLC column (Vydac). A series of 15-30 ul injections of crude peptides in 100% DMSO were used. Purity was checked using an analytical reverse phase HPLC column, with a diode-array spectrophotometer. One pass was adequate to give greater than 90% purity.
  • An exemplary fluorescent probe (Fig. 1 or Fig. 5) was designed to consist of the fluorescent moiety, 5-carboxyfluorescein, coupled to a pentapeptide ligand based on the known Src-SH2 high affinity tetrapeptide sequence derived from the core middle-T antigen.
  • the peptide ligand sequence is GpYEEI, containing the core middle-T antigen high-affinity Src-SH2 sequence (pYEEI), with an N-terminal glycine for ease of coupling.
  • 5-Carboxyfluorescein was chosen for several reasons. It is one of the few defined isomer fluoresceins available. It yields a conjugate with less flexibility than many available fluoresceins, which is important for minimizing the "propeller effect" that can interfere with FP based measurements and an activated version is commercially available (Molecular Probes, Inc.).
  • the probe sequence was prepared as follows: The peptide GpYEEI was coupled to the fluorescein moiety directly on the resin. The peptide sequence was assembled on Rink amide resin, and phosphorylated as described above. The resultant sequence was Fmoc-GpYEEI-RINK. The peptide/resin was deprotected with 20% piperidine in DMA, removing the FMOC protecting group, and leaving the free-amino terminus available for coupling. After thorough washing with DMA, 1.1 equivalent of 5-carboxyfluorescein succinimidyl ester (Molecular Probes) was added with 6 equivalents of diisopropylethylamine. Coupling was carried out for 1.5 hours, followed by 1 NMP and 3 DMA washes, and by a repeat coupling as above. The completed probe was cleaved and worked up as described above, yielding a probe termed FMT1.
  • the cells were lysed in 50 mM potassium phosphate, 250 mM NaCl, 5 mM DTT, 2 mM EDTA, 1 mm PMSF, pH 7.0 using a French pressure cell at 16,000 psi.
  • the protein was purified over carboxy-sulfon (J. T. Baker), a weak-strong cation exchanger.
  • the column was equilibrated with 50 mM potassium phosphate, 5 mM DTT, 0.02% NaN,, pH 7.0 and loaded with filtered bacterial lysate at 2 ml/minute.
  • the Src protein was eluted with a 1 M NaCl gradient.
  • the eluate was concentrated using a Centriprep 10 concentrator (Amicon, 10,000 MW cutoff) and centrifuged at 3000 x g.
  • the protein was then purified by gel filtration on a Sephacryl S-100 (Pharmacia) column equilibrated with 20 mM potassium phosphate, 50 mM NaCl, 5 mM DTT, 1 mM EDTA, 0.02% NaN réelle pH 7.4. Purity, as measured by SDS gel electrophoresis and RP-HPLC, is >95%.
  • the purified protein is stored frozen in 50 mM potassium phosphate, 500 mM NaCl, 10% glycerol, 5 mM DTT, 5 mM EDTA, 0.02% NaN 3 , pH 7.4.
  • Figs. 2A and 2B show that the affinity of the probe for the receptor domain is appropriate for conducting competitive binding assays and that saturable binding to a single site is observed, consistent with the assay of this invention and with competitive, reversible binding to a single site.
  • the designed probe FMT-1 has a Kd of -0.3 uM towards Src-SH2 in the standard buffer conditions. Some variation in observed Kd values will occur with changes in buffer conditions.
  • a Src competition assay (2% DMSO buffer) was conducted for the following tetrapeptides: Ac-pYEEI (open circle); Ac-pYpYEEI (closed square); Ac-pYGGL (plus sign); Ac-pYEDL (open triangle); Ac-DGVpYTGL (closed triangle).
  • Fig. 3 shows the competition curve plotting % probe binding vs. inhibitor concentration obtained in one set of experiments.
  • Standard (STD) buffer contains 20 mM phosphate (pH 7.4), 100 mM NaCl, 2 mM DTT, 1 mM EDTA, and 100 ug/ml BGG. Standard buffer is prepared by preparing NaCl, EDTA and phosphate stocks in Millipore water or the cleanest available water supply. The buffer is brought to volume in the same clean water.
  • the five buffers needed for these experiments are standard (STD) buffer, STD Buffer + 4% DMSO, 100% DMSO, STD buffer with labelled probe alone and STD buffer with Src protein and labelled probe.
  • One liter is made up of 100 ml of IM NaCl, 20 ml of IM phosphate (pH 7.4), 2 ml of 500 mM EDTA, 20 ml of 5 mg/ml BGG, and 858 ml of Clean Water.
  • the standard buffer is made up and stored at 4°C. Before each use, the DTT is added at 2 mM. This standard buffer is employed to make up the Standard Buffer + 4% DMSO stock also. This can be stored at 4°C as well and DTT can be added before use.
  • the SRC protein is used at a concentration of 0.75 uM final.
  • An example of SRC stock solution is 416.6 uM in STD buffer.
  • the peptide Ac-pYEEI is used at 100 uM final. It is desirable to make a 2X stock of this.
  • Fluor-GpYEEI is used as the probe at a concentration of 20 nM final.
  • the probe stock sent is 10 uM in STD buffer.
  • Each compound/peptidc and the control peptide is in separate tubes. They are at 2X stocks. It is desirable to make primary stock at 50 mM in 100% DMSO, then make the secondary stocks in STD Buffer + 4% DMSO at the appropriate concentration for the desired experiment.
  • a secondary stock is made by combining 2 mM stock in STD Buffer + 4% DMSO for one embodiment of an assay (Long Format, 2% DMSO). Another is 5 mM stock in 100% DMSO for Long Format assay with 20% DMSO. Another secondary stock solution is 400 uM stock in STD buffer + 4% DMSO for Short Format assay #1 and #2 -2% DMSO. 1 mM stock in 100% DMSO for Short Format #1 and #2 -20% DMSO
  • Compounds/peptides are used in singly. These examples are for a 1 :3 dilution but volumes may be changed to accommodate whatever dilution you wish.
  • This assay is run with 1/3 dilution steps with first well at 200 uM, 4 wells down, 22 cpds/plate, "landscape" orientation of plate with respect to Genesis deck, (i) 20% DMSO (ii) 2% DMSO
  • This assay is run with 1/2 dilution steps with first well at 1 mM, 10 wells down, 8 cpds/plate, "landscape" orientation of plate with respect to Genesis deck, (i) 20% DMSO (ii) 2% DMSO Dilution 5 Buffers:
  • the plate can be read between 5 and 30 minutes.
  • the assay has also been used for the proteins Zap, Syk and Lek. Those three proteins are produced analogously to the production of Src in E. coli, as described above with slight variations in production parameters such as salt concentration, DTT concentration, protein concentration, temperature and the like.
  • Src has a single SH2 domain
  • ZAP and Syk comprise two SH2 domains and the Lek protein comprises one SH2 domain and one SH3 domain.
  • the "first proteins” produced for these assays are produced generally as described for Src aa 145-251 in Example 3.
  • the proteins are represented in the table below.
  • the term “NC” means that the first protein contains both the N terminal and C terminal SH2 domains.
  • the sequences of Zap, Syk and Lek are known in the art. See also PCT/US96/13918 Table I
  • an exemplary probe for N,C-ZAP proteins i.e., proteins containing the two SH2 domains of human ZAP in series
  • an exemplary probe for N,C-Syk proteins i.e., proteins containing the two SH2 domains of human Syk in series
  • the SRC probe was also used with Lek.
  • the Lek protein has been assayed against fluorophore-labeled phosphopeptide ligand for the SH2 domain, fluorophore-labeled peptide ligand for the SH3 domain and fluorophore-labeled phosphopeptide double ligand for the SH2 and SH3 domains.
  • Figs. 2 A and 2B show that the affinity of the probe for the receptor domain is appropriate for conducting competitive binding assays and that saturable binding to a single site is observed, consistent with the assay of this invention and with competitive, reversible binding to a single site.
  • the data obtained from the saturation assay can, within the methods of the present invention, be used to assess whether a particular probe would be useful. For example, if a probe performs at a particular level in a saturation assay (e.g.,, Kd ⁇ 10uM and mP difference values >50 (difference in observed polarization in the presence and absence of protein)), it is indicative of its suitability for use in a competition assay of this invention.
  • Assay- General Proteins such as Src contain both an SH2 and an SH3 domain. Certain proteins can bind to such SH2-SH3 proteins not through SH2 or SH3 domains alone, but through both domains at once.
  • One example is the protein, pl30CAS, which is thought to bind Src via both SH2 and SH3 domains.
  • the SH3 and SH2 binding sequences of pl30CAS have been identified by deletional and site-specific mutagenesis (Nakamoto et. al., JBC 271 , 8959-8965, 1996). Residues 733-738 (RPLPSP) have been shown to be involved in binding (presumably to the SH3 domain), as has residue Tyr-762 (presumably phosphorylated and responsible for binding Src SH2). Fluorescent probes for FP assays were designed based on these sequences. Such probes can be used in assays that allow simultaneous screening for both SH2-specific and SH3-specific inhibitors.
  • FPC130 is the following:
  • Fluor represents 5-carboxyfluorescein conjugated through an amide bond to the peptide containing the sequence PLARRPLPPLP, which is specific for Src Sh3 domain binding (any other fluorescent probe might be substituted).
  • This probe had appropriate characteristics and an appropriate saturation curve with the Src protein to (e.g., appropriate Kd and total mP difference on protein binding) to serve as a probe for a competition assay.
  • a competition assay was developed for Src-SH2 (applicable to any fragment of Src containing the SH2 domain) with a non-fluorescein fluorophore.
  • the fluorophore is one of a family of fluorescent molecules containing the core fluorophore, 4,4-difluoro-4 bora-3a,4a-diaza-s-indacene (commercially available from Molecular Probes Inc.).
  • the structure of the fluorophore-peptide probe is:
  • the fluorophore is BODIPY-TRX, and has spectral characteristics very similar to the Texas Red family of fluorophores.
  • the use of a red fluorophore widens the utility of the assay, and permits one to screen test compounds that might have fluorescence characteristics similar to fluorescein itself.
  • This probe showed an adequate Kd and total mP difference on protein binding to be a probe for a competition assay.

Abstract

L'invention concerne un procédé de dosage in vitro permettant d'identifier une substance test qui inhibe l'association mutuelle d'une paire de protéines. Ce procédé consiste à produire une paire de protéines aptes à s'associer, l'une des protéins étant porteuse d'une substande fluorescente liée de manière covalente; à préparer un mélange contenant les deux protéins et au moins une substance test; à irradier le mélange avec de la lumière polarisée d'une longueur d'onde appropriée permettant l'excitation de la substance fluorescente telle que l'indique l'émission de lumière polarisée; à mesurer le degré de polarisation de l'émission et à déterminer l'effet dû à la présence ou à la concentration de la substance test en réduisant la polarisation de l'émission observée d'un mélange de deux protéines seules. L'activité inhibitrice de la substance test est en corrélation avec les valeurs réduites de dépolarisation.
PCT/US1997/006746 1996-04-18 1997-04-18 Dosage in vitro a polarisation de fluorescence WO1997039326A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP09530451A JP2000512737A (ja) 1996-04-18 1997-04-18 試験管内蛍光偏光検定法
AU28076/97A AU727108B2 (en) 1996-04-18 1997-04-18 In vitro fluorescence polarization assay
EP97922393A EP0898709A2 (fr) 1996-04-18 1997-04-18 DOSAGE $i(IN VITRO) A POLARISATION DE FLUORESCENCE

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US1559096P 1996-04-18 1996-04-18
US60/015,590 1996-04-18
US2987096P 1996-11-06 1996-11-06
US60/029,870 1996-11-06

Publications (2)

Publication Number Publication Date
WO1997039326A2 true WO1997039326A2 (fr) 1997-10-23
WO1997039326A3 WO1997039326A3 (fr) 1997-12-24

Family

ID=26687596

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/006746 WO1997039326A2 (fr) 1996-04-18 1997-04-18 Dosage in vitro a polarisation de fluorescence

Country Status (5)

Country Link
EP (1) EP0898709A2 (fr)
JP (1) JP2000512737A (fr)
AU (1) AU727108B2 (fr)
CA (1) CA2250067A1 (fr)
WO (1) WO1997039326A2 (fr)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0938586A1 (fr) * 1996-10-28 1999-09-01 Panvera Corporation Mesure de l'activite de la kinase par polarisation de fluorescence
EP0957365A1 (fr) * 1997-09-05 1999-11-17 Matsushita Electric Industrial Co., Ltd. Procede de polarisation de fluorescence
WO2001025477A2 (fr) * 1999-10-01 2001-04-12 Biofocus Discovery Limited Bioanalyse
WO2003004569A1 (fr) * 2001-07-02 2003-01-16 Arctic Diagnostics Oy Procede permettant d'ameliorer l'hydrophilicite de composes marqueurs fluorescents
WO2003005030A1 (fr) * 2001-07-02 2003-01-16 Arctic Diagnostics Oy Colorants de difluorure dipyrromethenebore absorbant deux photons et leurs applications
US7084141B2 (en) 2001-05-24 2006-08-01 Avanir Pharmaceuticals Inhibitors of macrophase migration inhibitory factor and methods for identifying the same
US7105307B2 (en) 1997-08-30 2006-09-12 Cyclacel, Ltd. Compositions and methods for screening for modulators of enzymatic activity
US7173036B2 (en) 2003-02-14 2007-02-06 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7361760B2 (en) 2003-08-22 2008-04-22 Avanir Pharmaceuticals Substituted naphthyridine derivatives as inhibitors of macrophage migration inhibitory factor and their use in the treatment of human diseases
US7365200B2 (en) 2005-03-24 2008-04-29 Avanir Pharmaceuticals Thienopyridinone derivatives as macrophage migration inhibitory factor inhibitors
WO2013056720A1 (fr) * 2011-10-18 2013-04-25 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Colorants fluorescents comportant des groupes hydroxyméthyle phosphorylés et leur utilisation en microscopie photonique et dans des techniques d'imagerie
US8487078B2 (en) 2003-07-29 2013-07-16 Life Technologies Corporation Kinase and phosphatase assays
WO2016008671A1 (fr) * 2014-07-15 2016-01-21 Valitacell Limited Procédé de mesure de concentration d'anticorps dans un échantillon
US10591463B2 (en) 2014-04-04 2020-03-17 Valitacell Limited Method of predicting phenotypic instability in a cell
US10626436B2 (en) 2015-04-01 2020-04-21 Valitacell Limited Method of determining a compositional or functional characteristic of a cell culture media

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4607950B2 (ja) * 2004-03-18 2011-01-05 トランス テック ファーマ，インコーポレイテッド 蛍光偏光アッセイ
US7608407B2 (en) * 2004-11-16 2009-10-27 Boehringer Ingelheim Pharmaceuticals, Inc. Fluorescence polarization assay for determining histidine decarboxylase activity

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4257774A (en) * 1979-07-16 1981-03-24 Meloy Laboratories, Inc. Intercalation inhibition assay for compounds that interact with DNA or RNA
EP0449269A1 (fr) * 1990-03-27 1991-10-02 Roche Diagnostics GmbH Méthode pour la détection des protéines contenant tyrosine phosphorylée
WO1993003377A1 (fr) * 1991-07-31 1993-02-18 Ziltener Hermann J Procede de detection de restes de phosphotyrosine
US5352660A (en) * 1991-10-31 1994-10-04 Mount Sinai Hospital Corporation Method for assaying for a substance that affects a SH2-phosphorylated ligand regulatory system
WO1997010253A1 (fr) * 1995-09-15 1997-03-20 Merck & Co., Inc. Dosage a rendement eleve utilisant des proteines hybrides

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4257774A (en) * 1979-07-16 1981-03-24 Meloy Laboratories, Inc. Intercalation inhibition assay for compounds that interact with DNA or RNA
EP0449269A1 (fr) * 1990-03-27 1991-10-02 Roche Diagnostics GmbH Méthode pour la détection des protéines contenant tyrosine phosphorylée
WO1993003377A1 (fr) * 1991-07-31 1993-02-18 Ziltener Hermann J Procede de detection de restes de phosphotyrosine
US5352660A (en) * 1991-10-31 1994-10-04 Mount Sinai Hospital Corporation Method for assaying for a substance that affects a SH2-phosphorylated ligand regulatory system
WO1997010253A1 (fr) * 1995-09-15 1997-03-20 Merck & Co., Inc. Dosage a rendement eleve utilisant des proteines hybrides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
B. A. LYNCH ET AL: "A fluorescence polarisation based Src-SH2 binding assay." ANALYTICAL BIOCHEMISTRY., vol. 247, no. 1, 1997, NEW YORK US, pages 77-82, XP002045666 *
P. WU ET AL: "A high-throughput STAT binding assay using fluorescence polatization." ANALYTICAL BIOCHEMISTRY., vol. 249, no. 1, 1997, NEW YORK US, pages 29-36, XP002045667 *

Cited By (41)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0938586A4 (fr) * 1996-10-28 2002-01-30 Panvera Corp Mesure de l'activite de la kinase par polarisation de fluorescence
EP0938586A1 (fr) * 1996-10-28 1999-09-01 Panvera Corporation Mesure de l'activite de la kinase par polarisation de fluorescence
US7105307B2 (en) 1997-08-30 2006-09-12 Cyclacel, Ltd. Compositions and methods for screening for modulators of enzymatic activity
EP0957365A1 (fr) * 1997-09-05 1999-11-17 Matsushita Electric Industrial Co., Ltd. Procede de polarisation de fluorescence
EP0957365A4 (fr) * 1997-09-05 2000-08-23 Matsushita Electric Ind Co Ltd Procede de polarisation de fluorescence
US6432632B2 (en) 1997-09-05 2002-08-13 Matsushita Electric Industrial Co., Ltd. Fluorescence polarization method
WO2001025477A2 (fr) * 1999-10-01 2001-04-12 Biofocus Discovery Limited Bioanalyse
WO2001025477A3 (fr) * 1999-10-01 2002-01-31 Biofocus Discovery Ltd Bioanalyse
US7235565B2 (en) 2001-05-24 2007-06-26 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7514225B2 (en) 2001-05-24 2009-04-07 Avanir Pharmaceuticals Method for screening an agent that modulates activity of macrophage migration inhibitory factor
US7105519B2 (en) 2001-05-24 2006-09-12 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7084141B2 (en) 2001-05-24 2006-08-01 Avanir Pharmaceuticals Inhibitors of macrophase migration inhibitory factor and methods for identifying the same
US7129236B2 (en) 2001-05-24 2006-10-31 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7157469B2 (en) 2001-05-24 2007-01-02 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7732146B2 (en) 2001-05-24 2010-06-08 Avanir Pharmaceuticals Method for screening an agent that modulates activity of macrophage migration inhibitory factor
US7192955B2 (en) 2001-05-24 2007-03-20 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7192961B2 (en) 2001-05-24 2007-03-20 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7432374B2 (en) 2001-05-24 2008-10-07 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7202248B2 (en) 2001-05-24 2007-04-10 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7230106B2 (en) 2001-05-24 2007-06-12 Avanir Pharmaceuticals Process for the preparation of inhibitors of macrophage migration inhibitory factor
US7435737B2 (en) 2001-05-24 2008-10-14 Avanir Pharmaceutials Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7238809B2 (en) 2001-05-24 2007-07-03 Avanir Pharmaceuticals Process for the preparation of inhibitors of macrophage migration inhibitory factor
US7763439B2 (en) 2001-07-02 2010-07-27 Arctic Diagnostics Oy Two-photon absorbing dipyrrometheneboron difluoride dyes and their applications
WO2003004569A1 (fr) * 2001-07-02 2003-01-16 Arctic Diagnostics Oy Procede permettant d'ameliorer l'hydrophilicite de composes marqueurs fluorescents
US7198958B2 (en) 2001-07-02 2007-04-03 Arctic Diagnostics Oy Method for increasing hydrophilicity of fluorescent label compounds
WO2003005030A1 (fr) * 2001-07-02 2003-01-16 Arctic Diagnostics Oy Colorants de difluorure dipyrromethenebore absorbant deux photons et leurs applications
US7173036B2 (en) 2003-02-14 2007-02-06 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7312221B2 (en) 2003-02-14 2007-12-25 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7312220B2 (en) 2003-02-14 2007-12-25 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7235546B2 (en) 2003-02-14 2007-06-26 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US8487078B2 (en) 2003-07-29 2013-07-16 Life Technologies Corporation Kinase and phosphatase assays
US7361760B2 (en) 2003-08-22 2008-04-22 Avanir Pharmaceuticals Substituted naphthyridine derivatives as inhibitors of macrophage migration inhibitory factor and their use in the treatment of human diseases
US7365200B2 (en) 2005-03-24 2008-04-29 Avanir Pharmaceuticals Thienopyridinone derivatives as macrophage migration inhibitory factor inhibitors
WO2013056720A1 (fr) * 2011-10-18 2013-04-25 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Colorants fluorescents comportant des groupes hydroxyméthyle phosphorylés et leur utilisation en microscopie photonique et dans des techniques d'imagerie
US9164086B2 (en) 2011-10-18 2015-10-20 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Fluorescent dyes with phosphorylated hydroxymethyl groups and their use in light microscopy and imaging techniques
US9523678B2 (en) 2011-10-18 2016-12-20 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Fluorescent dyes with phosphorylated hydroxymethyl groups and their use in light microscopy and imaging techniques
US10591463B2 (en) 2014-04-04 2020-03-17 Valitacell Limited Method of predicting phenotypic instability in a cell
WO2016008671A1 (fr) * 2014-07-15 2016-01-21 Valitacell Limited Procédé de mesure de concentration d'anticorps dans un échantillon
CN106662581A (zh) * 2014-07-15 2017-05-10 瓦里泰细胞有限公司 一种测定样品中抗体浓度的方法
EP3795999A1 (fr) * 2014-07-15 2021-03-24 Valitacell Limited Procédé permettant de déterminer l'abondance d'une molécule cible dans un échantillon
US10626436B2 (en) 2015-04-01 2020-04-21 Valitacell Limited Method of determining a compositional or functional characteristic of a cell culture media

Also Published As

Publication number Publication date
WO1997039326A3 (fr) 1997-12-24
CA2250067A1 (fr) 1997-10-23
EP0898709A2 (fr) 1999-03-03
JP2000512737A (ja) 2000-09-26
AU727108B2 (en) 2000-11-30
AU2807697A (en) 1997-11-07

Similar Documents

Publication Publication Date Title
US6207397B1 (en) In vitro fluorescence polarization assay
AU727108B2 (en) In vitro fluorescence polarization assay
US7341845B2 (en) Method for identifying kinesin inhibitors
US5693476A (en) Methods of screening for compounds capable of modulating vesicular release
JP2004523726A (ja) コンフォメーションに敏感な結合ペプチドの同定方法およびその利用
US8399201B2 (en) Polypeptide complex of TRPM8 and calmodulin and its uses thereof
Eto et al. Phosphorylation-induced conformational switching of CPI-17 produces a potent myosin phosphatase inhibitor
Moll et al. Biomolecular interaction analysis in functional proteomics
King et al. Solution structure of the chicken skeletal muscle troponin complex via small-angle neutron and X-ray scattering
Imran et al. Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions
US20040072319A1 (en) Molecules that modulate ubiquintin-dependent proteolysis and methods for identifying same
WO2003059943A2 (fr) Peptides specifiques a la conformation, de liaison de la proteine kinase, procedes et produits associes
US7807381B2 (en) Methods for assessing CDK5 activation and function
US20230374088A1 (en) PEPTIDE INHIBITORS OF GLIOMA-ASSOCIATED ONCOGENE DERIVED FROM THE COILED-COIL DIMERIZATION DOMAIN OF Kif7
Roth et al. Ubiquitin Binds to a Short Peptide Segment of Hydrolase UCH‐L3: A Study by FCS, RIfS, ITC and NMR
KR100888805B1 (ko) 특정 아미노산이 표지된 단백질과 1ｄ ｎｍｒ 기법을이용하여 단백질의 활성 부위에 결합하는 화합물을검색하는 방법
KR100456054B1 (ko) 특정 아미노산이 표지된 단백질과 2ｄ ｎｍｒ 기법을이용하여 단백질의 활성 부위에 결합하는 화합물을검색하는 방법
Imran WDR5 Network Analysis Using Ensemble Approaches
US7267959B2 (en) Drug target within the sulfonylurea receptor
Wong et al. Cardiac myosin-binding protein C N-terminal interactions with myosin and actin filaments: Opposite effects of phosphorylation and M-domain mutations
Hayes Chemical inhibitors of protein-protein interactions involved in G protein-mediated signaling events as potential therapeutics
WO2021150578A1 (fr) Biocapteurs de protéine f-actine marquée par fluorescence et procédés de recherche de médicament à haut débit
JP2003185655A (ja) プロリンリッチなペプチドとｓｈ３ドメイン含有ペプチドとの相互作用を阻害する化合物のスクリーニング法
Oy et al. ISB thanks the following sponsors for their very generous grants and funding support of our yearly meeting of the graduate school! The Academy of Finland The Ministry of Education
EP1107005A1 (fr) Méthode pour l'identification d'inhibiteurs de cytokinèse

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AU CA JP US US

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

AK Designated states

Kind code of ref document: A3

Designated state(s): AU CA JP US US

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase in:

Ref country code: CA

Ref document number: 2250067

Kind code of ref document: A

Format of ref document f/p: F

Ref document number: 2250067

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 1997922393

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1997922393

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1997922393

Country of ref document: EP