WO2001064939A2 - Process for obtaining inhibitors/activators of an enzyme - Google Patents

Process for obtaining inhibitors/activators of an enzyme Download PDF

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Publication number
WO2001064939A2
WO2001064939A2 PCT/EP2001/002438 EP0102438W WO0164939A2 WO 2001064939 A2 WO2001064939 A2 WO 2001064939A2 EP 0102438 W EP0102438 W EP 0102438W WO 0164939 A2 WO0164939 A2 WO 0164939A2
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Prior art keywords
enzyme
ptyr
thr
asp
arg
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PCT/EP2001/002438
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French (fr)
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WO2001064939A3 (en
Inventor
Yi Liu
Shaojie Wang
Zhong-Yin Zhang
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Morphochem Ag
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Priority to AU40673/01A priority Critical patent/AU4067301A/en
Publication of WO2001064939A2 publication Critical patent/WO2001064939A2/en
Publication of WO2001064939A3 publication Critical patent/WO2001064939A3/en

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    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • the present invention relates to a process for obtaining inhibitors/activators of an enzyme by using an enzymatically inactive mutant enzyme that binds substrate using a protein- protein interaction assay system.
  • the process for obtaining inhibitors/activators of an enzyme according to the invention by using a protein-protein interaction screening system such as a fluorescence polarization based assay, or an assay based on other methods such as SPA, (scintillation proximity assay) , confocal spectroscopy, surface plasmon resonance measuring assays (biacore) and HTRF (homogenous time resolved fluorescence) , is generally applicable.
  • SPA sintillation proximity assay
  • bias confocal spectroscopy
  • bias surface plasmon resonance measuring assays
  • HTRF homogenous time resolved fluorescence
  • PTPs Protein tyrosine phosphatases
  • PTP-IB Due to its negative regulator function on insulin signaling, PTP-IB has been implicated as a key mediator in the pathogenesis of insulin resistance and non-insulin-dependent dia- betes mellitus (NIDDM) . Therefore, potent and highly selective inhibitors of PTP1B are highly demanded. Studies on attempts to identify such inhibitors have been reported (Puius, et al., 1997; Taing, et . al . , 1999; robel, et . al . , 1999).
  • an object of the invention is to provide a generally applicable process for obtaining inhibitors/activators of an enzyme, and in particular to identify small molecules that inhibit PTP-IB and antagonize the attenuation of PTP-IB on the insulin signal.
  • the antagonists of PTP-IB can be used for potential anti-diabetic and anti-obesity drugs.
  • the invention provides a process for obtaining inhibitors/activators of an enzyme comprising the steps of a) contacting an enzymatically inactive but substrate binding mutant of an enzyme in the presence of a potential inhibitor/activator with a labeled substrate, b) measuring a physical property of the sample changed upon substrate binding, c) determining the level of inhibition/activation, e.g. by comparing with a standard curve, and, optionally, d) selecting said inhibitor/activator.
  • the enzyme is a protein tyrosine phosphatase, a kinase, such as a protein tyrosine kinase (TK) , or a protease or a Ras protein or a Raf protein.
  • TK protein tyrosine kinase
  • the changed and measured physical property of the sample is fluorescence polarization.
  • the invention further relates to protein tyrosine phosphatase inhibitors obtainable by the process according to the invention.
  • the invention further relates to the use of a protein tyrosine phosphatase inhibitor obtainable by a process according to the invention for the preparation of a medicament for treating diabetes or obesity.
  • the invention provides an assay for screening for inhibitors/activators of an enzyme and determining the level of said inhibition/activation comprising the steps of a) contacting an enzymatically inactive but substrate binding mutant of an enzyme in the presence of a potential inhibitor/activator with a labeled substrate, b) measuring a physical property of the sample changed upon substrate binding, c) determining the level of inhibition/activation, e.g. by comparing with a standard curve.
  • the enzyme is preferably a protein tyrosine phosphatase, a kinase, such as a protein tyrosine kinase (TK) , or a protease or a Ras protein or a Raf protein.
  • TK protein tyrosine kinase
  • the enzymes can be used in crude or purified form. Purification can be carried out in a per se usual manner.
  • the changed and measured physical property of the sample is fluorescence polarization.
  • Figure 1 to Figure 3 show the inactive PTP-lB/substrate bind- ing, dephosphorylated substrate binding, non-labeled peptide competition, and vanadate inhibition, measured by fluorescence polarization (FP) .
  • FP fluorescence polarization
  • Figure 4 shows the working scheme of the FP based assay.
  • Figure 5 shows the inactive PTPlB/substrate binding measured by FP as in Figure 1.
  • the substrate used is a fluorescein labeled dodecapeptide with all three tyrosine residues being phosphorylated.
  • Figure 6 shows the PTP1B binding competition from non-labeled peptide.
  • the FP measurement was performed the same as in Figure 1.
  • Figure 7 shows the PTP1B/C215S binding competition from known inhibitor 1 (Taing et al . ) using the monophosphorylated tyrosine peptide as the substrate.
  • Figure 8 shows the PTP1B/C215S binding competition from known inhibitor 1 (Taing et al . ) using the triphosphorylated tyrosine peptide as the substrate.
  • the cDNA encoding the catalytic domain of human PTPIB was obtained using PCR from a human fetal brain cDNA library (Stratagene) .
  • the PCR primers used were 5 ' -AGCTGGATCCATATGGAGATGGAAAAGGAGTT (encoding both a BamHI and a Ndel site), and 3 ' -ACGCGAATTCTTAATTGTGTGGCTCCAGGATTCG (encoding an EcoRI site) .
  • the PCR product was digested with BamHI and EcoRI and subcloned into a pUCll ⁇ vector.
  • the PTPIB coding sequence was confirmed by DNA sequencing.
  • the coding region for the PTPIB was cut from pUCll ⁇ -PTPIB with Ndel and EcoRI and ligated to the corresponding sites of plasmid pT7-7.
  • the MutaGene in vitro mutagenesis kit from Bio-Rad was used for carrying out site-directed mutagenesis.
  • the oligonucleotide primer used for C215S was 5' -TGGTGCACTCCAGTGCAGG-3 ' , where the underlined base indicates the change from the naturally occurring nucleotide.
  • the mutation was confirmed by DNA sequencing:
  • FP fluorescence polarization
  • FP is a well-known and powerful technique for the determination of molecular reactions in solution. If the molecules are small, their rotation and tumbling are faster and result in a random emitted light with respect to the plane of polarization (depolarization) , thus resulting in a low FP signal. However, if the small molecules bind to large molecules, their rotation and tumbling become slower and the emitted light will retain a proportional degree of polarization, thus resulting in a high FP signal. According to this principle, the inventors designed an inactive enzyme binding assay as the basis of the inventive process.
  • a fluorescine (Fluo) labeled phos- photyrosine peptide will be used as peptide substrate.
  • This dodecapeptide has the same sequence as the native substrate of PTP-IB in IR kinase domain.
  • a mutant form of PTP-IB for example PTP1B-C215S, will be used for the screen.
  • the crystal structures of a complex formed from this mutant (C215S) and a peptide substrate have shown that the mutant binds to the peptide substrate as the wild type enzyme (Barford, et al . , 1994; Jia, et . al . , 1995).
  • This assay is simple, fast, homogeneous, accurate, and works at low costs. It does not need the addition of antibody, which is a major advantage compared to conventional FP assays. It can be easily adapted to HTS in 96, 364, or 1536- well format.
  • the same general principle can be applied to any other target, such as kinases, e.g. protein tyrosine kinases, and proteases, or a Ras protein or a Raf protein.
  • This FP based inactive enzyme/peptide binding assay can be used to screen for agonists/activators or antagonists/inhibitors of any desired TK and protease.
  • the clone expressing inactivated PTP-IB enzyme was obtained from Zhong-Yin Zhang, Ph.D., Albert Einstein College of Medicine, Bronx, NY.
  • the protein was expressed and purified to homogeneity by the inventors.
  • the purified inactive PTPIB was confirmed by western blot using PTPIB antibody from Santa Cruz (California) .
  • the substrate used is preferably a fluorescine labeled dode- capeptide with one or three of the tyrosine residue being phosphorylated.
  • the amino acid sequence of the monophospho- rylated peptide is:
  • amino acid sequence of the triphosphorylated peptide is Fluo-Thr-Arg-Asp- Ile-pTyr-Glu-Thr-Asp-pTyr-pTyr-Arg-Lys -OH
  • Phosphorylated peptides are mentioned, e.g. in A. Salmeen et al., 2000, Molecular Cell, Vol. 6, 1401-1412.
  • Figure 1 shows the inactive PTP-lB/substrate binding measured by FP.
  • the fluopeptide concentration used was 1 nM.
  • Buffer contains 100 mM MES, pH6.5 , 1 mM EDTA, 1 mM DTT, 1 mM PMSF. Total measurement volume was 40 ul/well.
  • FP was meas- ured in a 96-well HE plate (LJL) after incubating the peptide with inactive enzyme at room temperature for 20 min.
  • Figure 2 shows the inactive PTP-IB will not bind the dephosphorylated peptide.
  • FpY was dephosphorylated by active GST- PTPIB.
  • the reaction mixtures which contain 40 nM FpY, 0.05 ⁇ g/ ⁇ l active GST-PTP1B (control FpY with no PTPIB) , 50 mM Tris, pH7.0, 0.1 mM CaCl 2 , 0.25 mg/ml BSA were incubated at 37C for 30 min.
  • the FP measurement was performed the same as in Figure 1.
  • Figure 3 shows the PTPlb binding competition from non-labeled peptide and inhibition from vanadate.
  • the FP measurement was performed the same as in Figure 1.
  • Figure 4 shows the working scheme of the FP based assay.
  • Figure 5 shows the inactive PTPlB/substrate binding measured by FP as in Figure 1.
  • the substrate used is a fluorescein labeled dodecapeptide with all three tyrosine residues being phosphorylated.
  • Figure 6 shows the PTPIB binding competition from non-labeled peptide. The FP measurement was performed the same as in Figure 1.
  • Figure 7 shows the PTP1B/C215S binding competition from known inhibitor 1 using the monophosphorylated tyrosine peptide as the substrate.
  • Figure 8 shows the PTP1B/C215S binding competition from known inhibitor 1 using the triphosphorylated tyrosine peptide as the substrate .
  • Taing M. , Keng, Y.F., Shen, K. , u, L., Lawrence, D.S., and

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Abstract

The invention relates of a generally applicable process for obtaining inhibitors/activators of an enzyme by using a mutant enzyme that binds but is enzymatically inactive using a fluorescence polarization based or other method to measure protein-protein interaction. The obtained inhibitors/activators can be used for the preparation of medicaments for treating diseases caused by or involved with the activity of the enzyme.

Description

Process for obtaining inhibitors/activators of an enzyme
The present invention relates to a process for obtaining inhibitors/activators of an enzyme by using an enzymatically inactive mutant enzyme that binds substrate using a protein- protein interaction assay system.
Introduction and background
The process for obtaining inhibitors/activators of an enzyme according to the invention by using a protein-protein interaction screening system, such as a fluorescence polarization based assay, or an assay based on other methods such as SPA, (scintillation proximity assay) , confocal spectroscopy, surface plasmon resonance measuring assays (biacore) and HTRF (homogenous time resolved fluorescence) , is generally applicable. However, the following description of the prior art and the invention will be focussed without any limitation to protein tyrosine phosphatase and fluorescence polarization as an example.
A reference list with more detailed bibliographic information is provided at the end of this description. Protein tyrosine phosphatases (PTPs) play critical roles in the regulation of various cellular processes. To date, a large number of PTPs has been identified. Among the superfamily of PTPs, PTP-IB was one of the earliest PTPs which has been cloned, sequenced, and characterized. Overexpression of PTP-IB inhibits ligand-stimulated receptor autophosphoryla- tion. Although PTP-IB was one of the first PTPases identified, the insight into the potential physiological function of this enzyme only came to light recently. PTP-IB has been shown to interact directly with the activated insulin receptor. It inactivates the insulin receptor (IR) by removing phosphate from tyrosine residues added by insulin and binding therefore acts in a negative feedback loop to down-regulate insulin signaling. Recent studies in mice lacking PTP-IB showed that the loss of PTP-IB activity causes enhanced insulin sensitivity and resistance to weight gain in mice (Elche- bly, et al . 1999). The findings suggested the possibility of treating type 2 diabetes with drugs that block PTP-IB activity. Therefore, PTP-IB can be a potential therapeutic target for treatment of type 2 diabetes and obesity.
Due to its negative regulator function on insulin signaling, PTP-IB has been implicated as a key mediator in the pathogenesis of insulin resistance and non-insulin-dependent dia- betes mellitus (NIDDM) . Therefore, potent and highly selective inhibitors of PTP1B are highly demanded. Studies on attempts to identify such inhibitors have been reported (Puius, et al., 1997; Taing, et . al . , 1999; robel, et . al . , 1999).
Thus, an object of the invention is to provide a generally applicable process for obtaining inhibitors/activators of an enzyme, and in particular to identify small molecules that inhibit PTP-IB and antagonize the attenuation of PTP-IB on the insulin signal. The antagonists of PTP-IB can be used for potential anti-diabetic and anti-obesity drugs.
These and other objects and features of the invention will be apparent from the description, drawings, and claims which follow.
Accordingly, the invention provides a process for obtaining inhibitors/activators of an enzyme comprising the steps of a) contacting an enzymatically inactive but substrate binding mutant of an enzyme in the presence of a potential inhibitor/activator with a labeled substrate, b) measuring a physical property of the sample changed upon substrate binding, c) determining the level of inhibition/activation, e.g. by comparing with a standard curve, and, optionally, d) selecting said inhibitor/activator.
Preferably the enzyme is a protein tyrosine phosphatase, a kinase, such as a protein tyrosine kinase (TK) , or a protease or a Ras protein or a Raf protein.
Preferably the changed and measured physical property of the sample is fluorescence polarization.
In one aspect the invention further relates to protein tyrosine phosphatase inhibitors obtainable by the process according to the invention.
In yet another aspect the invention further relates to the use of a protein tyrosine phosphatase inhibitor obtainable by a process according to the invention for the preparation of a medicament for treating diabetes or obesity.
In still another aspect the invention provides an assay for screening for inhibitors/activators of an enzyme and determining the level of said inhibition/activation comprising the steps of a) contacting an enzymatically inactive but substrate binding mutant of an enzyme in the presence of a potential inhibitor/activator with a labeled substrate, b) measuring a physical property of the sample changed upon substrate binding, c) determining the level of inhibition/activation, e.g. by comparing with a standard curve.
In the above assay the enzyme is preferably a protein tyrosine phosphatase, a kinase, such as a protein tyrosine kinase (TK) , or a protease or a Ras protein or a Raf protein. According to the invention the enzymes can be used in crude or purified form. Purification can be carried out in a per se usual manner.
Preferably the changed and measured physical property of the sample is fluorescence polarization.
Further advantageous and/or preferred embodiments of the invention are subject-matter of the subclaims .
Brief description of the drawings
The foregoing and other objects of the invention, the various features thereof, as well as the invention itself, may be more fully understood from the following description, when read together with the accompanying drawings, in which:
Figure 1 to Figure 3 show the inactive PTP-lB/substrate bind- ing, dephosphorylated substrate binding, non-labeled peptide competition, and vanadate inhibition, measured by fluorescence polarization (FP) .
Figure 4 shows the working scheme of the FP based assay.
Figure 5 shows the inactive PTPlB/substrate binding measured by FP as in Figure 1. Here, the substrate used is a fluorescein labeled dodecapeptide with all three tyrosine residues being phosphorylated.
Figure 6 shows the PTP1B binding competition from non-labeled peptide. The FP measurement was performed the same as in Figure 1.
Figure 7 shows the PTP1B/C215S binding competition from known inhibitor 1 (Taing et al . ) using the monophosphorylated tyrosine peptide as the substrate.
Figure 8 shows the PTP1B/C215S binding competition from known inhibitor 1 (Taing et al . ) using the triphosphorylated tyrosine peptide as the substrate.
Inhibitor 1 :
Figure imgf000006_0001
X = COCH2CH2p-C6H4CF2P03H2 In the following the invention is disclosed in more detail with reference to examples and to drawings. However, the described specific forms or preferred embodiments are to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the following description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Material and methods
I. Construction of PTPIB (Reference: Puius et al . (1997):
The cDNA encoding the catalytic domain of human PTPIB (amino acids 1-321) was obtained using PCR from a human fetal brain cDNA library (Stratagene) . The PCR primers used were 5 ' -AGCTGGATCCATATGGAGATGGAAAAGGAGTT (encoding both a BamHI and a Ndel site), and 3 ' -ACGCGAATTCTTAATTGTGTGGCTCCAGGATTCG (encoding an EcoRI site) . The PCR product was digested with BamHI and EcoRI and subcloned into a pUCllδ vector. The PTPIB coding sequence was confirmed by DNA sequencing. The coding region for the PTPIB was cut from pUCllδ-PTPIB with Ndel and EcoRI and ligated to the corresponding sites of plasmid pT7-7.
II. Construction of the C215S Mutant of PTPIB (Reference: Puius et al . (1997) :
The MutaGene in vitro mutagenesis kit from Bio-Rad was used for carrying out site-directed mutagenesis. The oligonucleotide primer used for C215S was 5' -TGGTGCACTCCAGTGCAGG-3 ' , where the underlined base indicates the change from the naturally occurring nucleotide. The mutation was confirmed by DNA sequencing:
Human Protein Tyrosine phosphatase IB (PTP1B/C215S) : 1-321 amino acids
ATG GAG ATG GAA AAG GAG TTC GAG CAG ATC GAC AAG TCC GGG AGC TGG GCG GCC ATT TAC M E M E K E F E Q I D K S G S W A A I Y 20
CAG GAT ATC CGA CAT GAA GCC AGT GAC TTC CCA TGT AGA GTG GCC AAG CTT CCT AAG AAC Q D I R H E A S D F P C R V A K L P K N 40
AAA AAC CGA AAT AGG TAC AGA GAC GTC AGT CCC TTT GAC CAT AGT CGG ATT AAA CTA CAT K N R N R Y R D V S P F D H S R I K L H 60
CAA GAA GAT AAT GAC TAT ATC AAC GCT AGT TTG ATA AAA ATG GAA GAA GCC CAA AGG AGT Q E D N D Y I N A S L I K M E E A Q R S 80 TAC ATT CTT ACC CAG GGC CCT TTG CCT AAC ACA TGC GGT CAC TTT TGG GAG ATG GTG TGG
Y I L T Q G P L P N T C G H F W E M V W 100
GAG CAG AAA AGC AGG GGT GTC GTC ATG CTC AAC AGA GTG ATG GAG AAA GGT TCG TTA AAA E Q K S R G V V M L N R V M E K G S L K 120
TGC GCA CAA TAC TGG CCA CAA AAA GAA GAA AAA GAG ATG ATC TTT GAA GAC ACA AAT TTG C A Q Y W P Q K E E K E M I F E D T N L 140
AAA TTA ACA TTG ATC TCT GAA GAT ATC AAG TCA TAT TAT ACA GTG CGA CAG CTA GAA TTG K L T L I S E D I K S Y Y T V R Q L E L 160
GAA AAC CTT ACG ACC CAA GAA ACT CGA GAG ATC TTA CAT TTC CAC TAT ACC ACA TGG CCT
E N L T T Q E T R E I L H F H Y T T W P 180 GAC TTT GGA GTC CCT GAA TCA CCA GCC TCA TTC TTG AAC TTT CTT TTC AAA GTC CGA GAG
D F G V P E S P A S F L N F L F K V R E 200
TCA GGG TCA CTC AGC CCG GAG CAC GGG CCC GTT GTG GTG CAC TCC AGT GCA GGC ATC GGC S G S L S P E H G P V V V H S S A G I G 220
AGG TCT GGA ACC TTC TGT CTG GCT GAT ACC TGC CTC CTG CTG ATG GAC AAG AGG AAA GAC R S G T F C L A D T C L L L M D K R K D 240
CCT TCT TCC GTT GAT ATC AAG AAA GTG CTG TTA GAA ATG AGG AAG TTT CGG ATG GGG TTG P S S V D I K K V L L E M R K F R M G L 260
ATC CAG ACA GCC GAC CAG CTG CGC TTC TCC TAC CTG GCT GTG ATC GAA GGT GCCAAA TTC
I Q T A D Q L R F S Y L A V I E G A K F 280 ATC ATG GGG GAC TCT TCC GTG CAG GAT CAG TGG AAG GAG CTT TCC CAC GAG GAC CTG GAG
I M G D S S V Q D Q W K E L S H E D L E 300
CCC CCA CCC GAG CAT ATC CCC CCA CCT CCC CGG CCA CCC AAA CGA ATC CTG GAG CCA CAC P P P E H I P P P P R P P K R I L E P H 320
AAT
N III. Assay design
According to the invention and for its exemplification a fluorescence polarization (FP) based assay has been developed for PTP-IB screening as an example. FP is a well-known and powerful technique for the determination of molecular reactions in solution. If the molecules are small, their rotation and tumbling are faster and result in a random emitted light with respect to the plane of polarization (depolarization) , thus resulting in a low FP signal. However, if the small molecules bind to large molecules, their rotation and tumbling become slower and the emitted light will retain a proportional degree of polarization, thus resulting in a high FP signal. According to this principle, the inventors designed an inactive enzyme binding assay as the basis of the inventive process.
In this assay, for example a fluorescine (Fluo) labeled phos- photyrosine peptide will be used as peptide substrate. This dodecapeptide has the same sequence as the native substrate of PTP-IB in IR kinase domain. Instead of the active enzyme, a mutant form of PTP-IB, for example PTP1B-C215S, will be used for the screen. The crystal structures of a complex formed from this mutant (C215S) and a peptide substrate have shown that the mutant binds to the peptide substrate as the wild type enzyme (Barford, et al . , 1994; Jia, et . al . , 1995). However, due to lack of the catalytic activity, it cannot dephosphorylate the peptide. Thus, the phospho-peptide will bind to the inactive enzyme and form a peptide/protein com- plex resulting in a change of the FP signal. If a compound is an inhibitor/antagonist of PTP-IB, it will inhibit pep- tide/inactive enzyme binding. Therefore, samples that show lower degrees of depolarization will be identified as inhibi- tors/antagonists of PTP-IB. The working scheme of this assay is shown in Figure 3.
This assay is simple, fast, homogeneous, accurate, and works at low costs. It does not need the addition of antibody, which is a major advantage compared to conventional FP assays. It can be easily adapted to HTS in 96, 364, or 1536- well format. The same general principle can be applied to any other target, such as kinases, e.g. protein tyrosine kinases, and proteases, or a Ras protein or a Raf protein. This FP based inactive enzyme/peptide binding assay can be used to screen for agonists/activators or antagonists/inhibitors of any desired TK and protease.
Results
The clone expressing inactivated PTP-IB enzyme was obtained from Zhong-Yin Zhang, Ph.D., Albert Einstein College of Medicine, Bronx, NY. The protein was expressed and purified to homogeneity by the inventors. The purified inactive PTPIB was confirmed by western blot using PTPIB antibody from Santa Cruz (California) .
The substrate used is preferably a fluorescine labeled dode- capeptide with one or three of the tyrosine residue being phosphorylated. The amino acid sequence of the monophospho- rylated peptide is:
Fluo-Thr-Arg-Asp-Ile-pTyr-Glu-Thr-Asp-Tyr-Tyr-Arg-Lys-OH
The amino acid sequence of the triphosphorylated peptide is Fluo-Thr-Arg-Asp- Ile-pTyr-Glu-Thr-Asp-pTyr-pTyr-Arg-Lys -OH
Phosphorylated peptides are mentioned, e.g. in A. Salmeen et al., 2000, Molecular Cell, Vol. 6, 1401-1412.
Figure 1 shows the inactive PTP-lB/substrate binding measured by FP. Here, the fluopeptide concentration used was 1 nM. Buffer contains 100 mM MES, pH6.5 , 1 mM EDTA, 1 mM DTT, 1 mM PMSF. Total measurement volume was 40 ul/well. FP was meas- ured in a 96-well HE plate (LJL) after incubating the peptide with inactive enzyme at room temperature for 20 min.
Figure 2 shows the inactive PTP-IB will not bind the dephosphorylated peptide. FpY was dephosphorylated by active GST- PTPIB. The reaction mixtures, which contain 40 nM FpY, 0.05 μg/μl active GST-PTP1B (control FpY with no PTPIB) , 50 mM Tris, pH7.0, 0.1 mM CaCl2, 0.25 mg/ml BSA were incubated at 37C for 30 min. The FP measurement was performed the same as in Figure 1.
Figure 3 shows the PTPlb binding competition from non-labeled peptide and inhibition from vanadate. The FP measurement was performed the same as in Figure 1.
Figure 4 shows the working scheme of the FP based assay.
Figure 5 shows the inactive PTPlB/substrate binding measured by FP as in Figure 1. Here, the substrate used is a fluorescein labeled dodecapeptide with all three tyrosine residues being phosphorylated. Figure 6 shows the PTPIB binding competition from non-labeled peptide. The FP measurement was performed the same as in Figure 1.
Figure 7 shows the PTP1B/C215S binding competition from known inhibitor 1 using the monophosphorylated tyrosine peptide as the substrate.
Figure 8 shows the PTP1B/C215S binding competition from known inhibitor 1 using the triphosphorylated tyrosine peptide as the substrate .
References
Barford, D., Flint, A.J., Tonks, N.K., 1994, Science, 263:1397-1404
Elchebly, M, et . al . , and Kennedy, B.P., 1999, Science, 283, 1544-1546
Jia, Z., Barford, D., Flint, A.J., and Tonks, N.K., 1995, Science, 268:1544-1548
Puius, Y. , Zhao, Y. , Sullivan, M. , Lawrence, D., Almo., S., and Zhang, Z., 1997., PNAS, 94:13420-13425
Taing, M. , Keng, Y.F., Shen, K. , u, L., Lawrence, D.S., and
Zhang, Z., 1999, Biochemistry, 38:3793-3803 robel, J., Sredy, J. , Moxham, C, et al . , and Zhang, Z., 1999, J. Med. Chem., 42:3199-3202

Claims

Claims
Process for obtaining inhibitors/activators of an enzyme comprising the steps of a) contacting an enzymatically inactive but substrate binding mutant of an enzyme in the presence of a potential inhibitor/activator with a labeled substrate, b) measuring a physical property of the enzyme changed upon substrate binding, c) determining the level of inhibition/activation, and d) optionally, selecting said inhibitor/activator.
Process according to claim 1 wherein said enzyme is a protein tyrosine phosphatase, a kinase, such as a pro- tein tyrosine kinase, or a protease or a Ras protein or a Raf protein.
Process according to claim 1 or 2 wherein said enzyme is protein tyrosine phosphatase (PTP) IB and said mutant thereof is PTP-1B-C215S .
Process according to claim 3 wherein said labeled substrate is Fluo-Thr-Arg-Asp-Ile-pTyr-Glu-Thr-Asp-Tyr-Tyr-Arg-Lys-OH wherein Fluo means a fluorescent residue, and said changed and measured physical property of the sample is fluorescence polarization.
Process according to claim 3 wherein said labeled sub- strate is
Fluo-Thr-Arg-Asp-Ile-pTyr-Glu-Thr-Asp-pTyr-pTyr-Arg-Lys-OH wherein Fluo means a fluorescent residue, and said changed and measured physical property of the sample is fluorescence polarization.
Protein tyrosine phosphatase inhibitor obtainable by a process as defined in anyone of claims 3 to 5.
Use of a protein tyrosine phosphatase inhibitor obtainable by a process as defined in anyone of claims 3 to 5 for the preparation of a medicament for treating diabetes .
Use of a protein tyrosine phosphatase inhibitor obtained by a process as defined in anyone of claims 3 to 5 for the preparation of a medicament for treating obesity.
Assay for screening for inhibitors/activators of an enzyme and determining the level of said inhibition/acti- vation comprising the steps of a) contacting an enzymatically inactive but substrate binding mutant of an enzyme in the presence of a potential inhibitor/activator with a labeled substrate, b) measuring a physical property of the enzyme changed upon substrate binding, and c) determining the level of inhibition/activation.
Assay according to claim 9 wherein said enzyme is a protein tyrosine phosphatase, a protein tyrosine kinase or a protease.
11. Assay according to claim 9 or 10 wherein said enzyme is protein tyrosine phosphatase (PTP) IB and said mutant thereof is PTP-1B-C215S .
12. Process according to claim 11 wherein said labeled substrate is
Fluo-Thr-Arg-Asp-Ile-pTyr-Glu-Thr-Asp-Tyr-Tyr-Arg-Lys-OH, wherein Fluo means a fluorescent residue, and said changed and measured physical property of the sample is fluorescence polarization.
13. Process according to claim 11 wherein said labeled substrate is
Fluo-Thr-Arg-Asp-Ile-pTyr-Glu-Thr-Asp-pTyr-pTyr-Arg-Lys-OH wherein Fluo means a fluorescent residue, and said changed and measured physical property of the sample is fluorescence polarization.
PCT/EP2001/002438 2000-03-02 2001-03-02 Process for obtaining inhibitors/activators of an enzyme WO2001064939A2 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998018956A1 (en) * 1996-10-28 1998-05-07 Panvera Corporation Kinase activity measurement using fluorescence polarization
WO2001061031A2 (en) * 2000-02-14 2001-08-23 Ceptyr, Inc. Improved assay for protein tyrosine phosphatases

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998018956A1 (en) * 1996-10-28 1998-05-07 Panvera Corporation Kinase activity measurement using fluorescence polarization
WO2001061031A2 (en) * 2000-02-14 2001-08-23 Ceptyr, Inc. Improved assay for protein tyrosine phosphatases

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
TAING M ET AL: "Potent and highly selective inhibitors of the protein tyrosine phosphatase 1B" BIOCHEMISTRY, AMERICAN CHEMICAL SOCIETY. EASTON, PA, US, vol. 38, no. 12, 23 March 1999 (1999-03-23), pages 3793-3803, XP002163526 ISSN: 0006-2960 cited in the application *
WROBEL JAY ET AL: "PTP1B inhibition and antihyperglycemic activity in the ob/ob mouse model of novel 11-arylbenzo(b)naphtho(2,3-d)furans and 11-arylbenzo(b)naphtho(2,3-d)thiophenes." JOURNAL OF MEDICINAL CHEMISTRY, vol. 42, no. 17, 26 August 1999 (1999-08-26), pages 3199-3202, XP002181876 ISSN: 0022-2623 cited in the application *
ZHANG YAN-LING ET AL: "Suramin is an active site-directed, reversible, and tight-binding inhibitor of protein-tyrosine phosphatases." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 20, 15 May 1998 (1998-05-15), pages 12281-12287, XP002181875 ISSN: 0021-9258 *

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