Method for the screening of compounds that inhibit the interaction between a proline-rich peptide and a SH3 domain comprising peptide
First Claim
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1. A method for the screening of a candidate compound for inhibiting the interaction between a proline-rich peptide and a SH3 domain-comprising peptide by Homogeneous Time-Resolved Fluorescence technique, said method comprising the steps of:
- a) providing a buffer solution containing a proline-rich peptide which is labelled with a first fluorescent marker, wherein said first fluorescent marker is the first partner or the second partner of paired FRET fluorescence markers;
b) adding the candidate compound to the solution obtained at step a);
c) adding the SH3 domain-comprising peptide which is directly or indirectly labelled with a second fluorescent marker and wherein said second fluorescent marker is the second partner or the first partner of paired FRET fluorescence markers;
d) submitting the mixture obtained at step d) to a source of energy at a wavelength corresponding to the excitation wavelength of the first partner of paired FRET fluorescence markers and measuring the fluorescence signal at the emission wavelength of the second partner of paired FRET fluorescence markers;
e) comparing the fluorescence signal value obtained at step e) with the fluorescence signal value obtained when step b) is omitted to determine if the candidate compound inhibits the interaction between the proline-rich peptide and the SH3 domain-comprising peptide.
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Abstract
The present invention relates to a method for the screening of compounds of therapeutical value which possess the ability of inhibiting the interaction between a proline-rich peptide and a SH3 domain-comprising peptide, which compounds are able to selectively target proteins involved in a variety of intracellular signalling pathways.
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Citations
34 Claims
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1. A method for the screening of a candidate compound for inhibiting the interaction between a proline-rich peptide and a SH3 domain-comprising peptide by Homogeneous Time-Resolved Fluorescence technique, said method comprising the steps of:
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a) providing a buffer solution containing a proline-rich peptide which is labelled with a first fluorescent marker, wherein said first fluorescent marker is the first partner or the second partner of paired FRET fluorescence markers;
b) adding the candidate compound to the solution obtained at step a);
c) adding the SH3 domain-comprising peptide which is directly or indirectly labelled with a second fluorescent marker and wherein said second fluorescent marker is the second partner or the first partner of paired FRET fluorescence markers;
d) submitting the mixture obtained at step d) to a source of energy at a wavelength corresponding to the excitation wavelength of the first partner of paired FRET fluorescence markers and measuring the fluorescence signal at the emission wavelength of the second partner of paired FRET fluorescence markers;
e) comparing the fluorescence signal value obtained at step e) with the fluorescence signal value obtained when step b) is omitted to determine if the candidate compound inhibits the interaction between the proline-rich peptide and the SH3 domain-comprising peptide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 34)
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- 24. A screening reagent consisting of a proline-rich peptide which is labelled with a first fluorescent marker, wherein said first fluorescent marker is the first partner or the second partner of paired FRET fluorescence markers.
- 27. A screening reagent consisting of a SH3 domain-comprising peptide which is directly or indirectly labelled with a second fluorescent marker and wherein said second fluorescent marker is the second partner or the first partner of paired FRET fluorescence markers.
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29. A kit for the screening of a candidate compound for inhibiting the interaction between a proline-rich peptide and a SH3 domain-comprising peptide comprising:
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a) a first screening reagent consisting of a proline-rich peptide which is labelled with a first fluorescence marker, wherein said first fluorescent marker is the first partner or the second partner of paired FRET fluorescence markers;
b) a second screening reagent consisting of a SH3 domain-comprising peptide which is directly or indirectly labelled with a second fluorescent marker and wherein said fluorescent marker is the second partner or the first partner of paired FRET fluorescence markers.
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31. A complex formed between:
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a) a proline-rich peptide which is labelled with a first fluorescent marker, wherein said first fluorescent marker is the first partner or the second partner of paired FRET fluorescence markers; and
b) a SH3 domain-comprising peptide which is directly or indirectly labelled with a second fluorescent marker and wherein said second fluorescent marker is the second partner or the first partner of paired FRET fluorescence markers.
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32. A complex formed between:
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a) a proline-rich peptide which is labelled with a first fluorescent marker, wherein said first fluorescent marker is the first partner or the second partner of paired FRET fluorescence markers; and
b) a candidate compound which inhibits the interaction between the proline-rich peptide defined in a) and a SH3 domain-comprising peptide.
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33. A complex formed between:
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a) a SH3 domain-comprising peptide which is directly or indirectly labelled with a second fluorescent marker and wherein said second fluorescent marker is the second partner or the first partner of paired FRET fluorescence markers; and
b) a candidate compound which inhibits the interaction between a proline-rich peptide and the SH3 domain-comprising peptide defined in a).
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Specification