Assays and kits for detecting protein binding
View Patent ↗The invention provides methods for determining the interactions between phage-displayed proteins and test molecules. The phage-displayed proteins are contacted with a reference moiety in the presence and absence of a test molecule; the behavior of the phage-displayed proteins as a function of concentration of the test molecule permits calculation of the binding affinity of the phage-displayed protein for the test molecule.
1. A method for identifying a test molecule that binds to a phage-displayed polypeptide, wherein the test molecule is not previously known to bind to the phage-displayed polypeptide, the method comprising:
(a) contacting a reference moiety with the phage-displayed polypeptide in the presence and absence of test molecule, wherein the reference moiety is immobilized on a solid support, wherein the reference moiety is known to bind to the phage-displayed polypeptide and further wherein the phage-displayed polypeptide is a kinase polypeptide, and reference moiety binds to an ATP site of the kinase polypeptide;
(b) further wherein (i) binding of the phage-displayed polypeptide to the reference moiety occurs if the test molecule does not bind to the phage-displayed polypeptide, and wherein (ii) non-binding of the phage-displayed polypeptide to the reference moiety occurs if the test molecule does bind to the phage-displayed polypeptide;
(c) removing unbound phage-displayed polypeptide; and
(d) detecting phage-displayed polypeptide bound to the immobilized reference moiety in step (b);
whereby a decrease in an amount of phage-displayed polypeptide bound to the immobilized reference moiety contacted in the presence of the test molecule as compared to an amount of phage-displayed polypeptide bound to the immobilized reference moiety in the absence of the test molecule identifies the test molecule as a binder of the phage-displayed polypeptide.
2. The method of claim 1 , wherein the presence of the phage displaying the phage-displayed polypeptide is detected by quantitative polymerase chain reaction (qPCR).
3. The method of claim 1 , further comprising quantifying the amount of phage displaying the phage-displayed polypeptide bound to the immobilized reference moiety.
4. The method of claim 3 , wherein the quantifying is by qPCR, phage plaque assay or fluorescence polarization.
5. The method of claim 1 , wherein the reference moiety is purvalanol B, SU5402, SU6668, PD-173955, SB202190, staurosporine, SB203580, VX-745, BIRB-796, CC-401, imatinib mesylate, gefitinib, erlotinib, CI-1033, GW-2016, EKB-569, ZD-6474, vatalanib/PTK-787, SU11248, MLN-518, roscovitine/CYC202, or flavopiridol.
6. The method of claim 1 , wherein the protein kinase polypeptide is MAPKI2, MAP2K6, GPRK7, CDK7, CDK9, PCTK1, JNK1α/MAPK8α, JNK1β3/MAPK8β, JNK2a/MAPK9a, JNK3/MAPK10, CDK2, DAPK2, DMPK, NEK2, PAM, PAK6, KIAA1048, STK16, RS6KA2, RS6KA3, RS6KA5, LCK, PRKAA2, CSK, DAPK3, PRKACA, murine PAM, PAK3, PAK7, BIKE, murine STK3, STK4, STKI5/STK6(BTAK)/AURORA2, PIMI, PIM2, CAMK1, LOC57118 (CamK1-like), CamKIG, CamK2A, CamK2B, CamK2D, CamK2G, CamKKI, CamKK2, FGFR1, PDGFRβ, casein kinase 1, gamma 1, MAPK14/p38, STK18, STK25, VEGFR2, ABL, ABL mutants, BRAF, BRAF mutants, EGFR, EGFR mutants, ERBB2, ERBB4, p38α, FLT3, CDK1, or CDK4.
7. The method of claim 1 , further comprising determining the concentration of the test molecule at which about 50% of the phage-displayed polypeptide are bound to the reference moiety relative to the amount bound in the absence of the test molecule, wherein the concentration at which 50% of the phage-displayed polypeptide are bound to the reference moiety is the value of the dissociation constant (K d ) of the phage-displayed polypeptide.
8. The method of claim 1 , wherein the solid support is a bead.
9. The method of claim 8 , wherein the bead is in a column or a well of a multi-well plate.
10. The method of claim 1 , wherein the solid support is a well of a multi-well plate.
11. A method for identifying whether a test molecule binds to more than one member of a group of different phage-displayed polypeptides, wherein the test molecule is not previously known to bind to more than one member of the group of phage-displayed polypeptides, the method comprising:
(a) contacting, in a single well, a group of different phage-displayed polypeptides with a reference moiety in the presence and absence of a test molecule, wherein the reference moiety is immobilized on a solid support and wherein the reference moiety is known to bind to the group of phage-displayed polypeptides, said polypeptides being phage-displayed protein kinases and the reference moiety binding to an ATP site of each phage-displayed protein kinase polypeptide, and wherein the test molecule is tested for binding to one or more members of the group of phage-displayed polypeptides;
(b) further wherein (i) binding of a phage-displayed polypeptide to the reference moiety occurs if the test molecule does not bind to the phage-displayed polypeptide, and wherein (ii) binding of the phage-displayed polypeptide to the reference moiety does not occur if the test molecule does bind to the phage-displayed polypeptide;
(c) removing the unbound phage-displayed polypeptides from a mixture of step (b); and
(d) detecting the presence of each member of the group of phage-displayed polypeptides bound to the immobilized reference moiety after removing the unbound phage-displayed polypeptides;
whereby decreases in the amounts of binding of more than one member of the group of phage-displayed polypeptides to the immobilized reference moiety in the presence of the test molecule as compared to the absence of the test molecule identifies the test molecule as a binder of more than one member of the group of phage-displayed polypeptides.
12. The method of claim 11 , wherein the presence of each member of the group of phage-displayed polypeptides is detected by qPCR.
13. The method of claim 11 , further comprising quantifying the amount of each member of the group of phage-displayed polypeptides bound to the immobilized reference moiety.
14. The method of claim 13 , wherein the quantifying is by qPCR, phage plaque assay or fluorescence polarization.
15. The method of claim 11 , wherein the reference moiety is purvalanol B, SU5402, SU6668, PD-173955, SB202190, staurosporine, SB203580, VX-745, BIRB-796, CC-401, imatinib mesylate, gefitinib, erlotinib, CI-1033, GW-2016, EKB-569, ZD-6474, vatalanib/PTK-787, SU 11248, MLN-518, roscovitine/CYC202, or flavopiridol.
16. The method of claim 11 , wherein the protein kinase polypeptides are independently selected from the group consisting of MAPK12, MAP2K6, GPRK7, CDK7, CDK9, PCTK1, JNK, JNK1α/MAPK8α, JNKIβ3/MAPK8β, JNK2a/MAPK9a, JNK3/MAPK10, CDK2, DAPK2, DMPK, NEK2, PAM, PAK6, KIAA1048, STK16, RS6KA2, RS6KA3, RS6KA5, LCK, PRKAA2, CSK, DAPK3, PRKACA, murine PAM, PAK3, PAK7, BTKE, murine STK3, STK4, STK15/STK6(BTAK)/AURORA2, PIMI, PIM2, CAMK1, LOC57118 (CamK1-like), CamKIG, CamK2A, CamK2B, CamK2D, CamK2G, CamKKI, CamKK2, FGFR1, PDGFRβ, casein kinase 1, gamma 1, MAPK14/p38, STKI8, STK25, VEGFR2, ABL, ABL mutants, BRAF, BRAF mutants, EGFR, EGFR mutants, ERBB2, ERBB4, p38α, FLT3, CDK1, and CDK4.
17. The method of claim 11 , wherein the solid support is a bead.
18. The method of claim 17 , wherein the bead is in a column or a well of a multi-well plate.
19. The method of claim 11 , wherein the solid support is a well of a multi-well plate.
20. The method of claim 1 , wherein the reference moiety is a small molecule.
21. The method of claim 11 , wherein the reference moiety is a small molecule.
22. The method of claim 1 , wherein said phage-displayed polypeptide is present as a homogeneous group of phage-displayed polypeptides.