EP0770876A1
Expired / Lapsed
EP
⏱️ 0.0 years remaining
A screening method for identifying ligands for target proteins
Assignee: Anadys Pharmaceuticals Inc
•
Filing: 1996-10-17
•
Granted: —
•
Expires: 2016-10-17
Abstract
Abstract A novel method for screening chemical compounds (test ligands) for potential pharmaceutical effectiveness is provided. The disclosed method identifies possible therapeutic test ligands by placing them in the presence of target proteins and determining their ability to increase or decrease the ratio of folded target protein to unfolded target protein. The present methods do not require that biochemical function of the target protein be known, nor that any other ligands be previously identified.
Inventors
- 👤 Andrew Pakula
- 👤 James Bowie
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-24 15:59
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/34; C12Q1/37; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/68
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
194 words
A method for rapid, large-scale screening to identify a ligand that binds to a predetermined target protein, comprising the steps of: (a) selecting as test ligands a plurality of compounds not known to bind to the target protein; (b) incubating each of said test ligands and the target protein to produce a test combination; (c) incubating the target proten in the absence of a test ligand to produce a control combination; (d) treating the test and control combinations to cause the target protein in the control combination to unfold to a measurable extent; (e) determining the extent to which the target protein occurs in the folded state, the unfolded state or both in the test combination and in the control combination; (f) comparing the determination made in step (e) between the test and control combinations, wherein if the target protein is present in the folded state to a greater extent in the test combination than in the control combination, the test ligand is a ligand that binds to the target protein; and (g) repeating steps (b)-(f) with a plurality of said test ligands until a ligand that binds to the target protein is identified.
Claim 2
Dependent Claim
23 words
The method of claim 1, wherein the treating step comprises at least one of altering the temperature, adding denaturing compounds, and combinations thereof.
Claim 3
Dependent Claim
33 words
The method of claim 2, wherein the treating step comprises adding denaturing compounds and the denaturing compounds are selected from the group consisting of urea, guanidinium and salts thereof, detergents, and combinations thereof.
Claim 4
Dependent Claim
36 words
A method according to claim 1 or 2, wherein the treating step includes subjecting the test and control combinations to increased temperature, thereby causing a detectable fraction of said target to exist in an unfolded state.
Claim 5
Independent Claim
190 words
A method for rapid, large-scale screening to identify a ligand that binds to a predetermined target protein, comprising the steps of: (a) selecting as test ligands a plurality of compounds not known to bind to the target protein; (b) incubating each of said test ligands and the target protein under conditions appropriate for the target protein to unfold to a measurable extent, thereby producing a test combination; (c) incubating the target protein as in step (b), but in the absence of a test ligand, to produce a control combination; (d) determining the extent to which the target protein occurs in the folded state, the unfolded state or both in the test combination and in the control combination; (e) comparing the determination made in step (d) between the test and control combinations, wherein if the target protein is present in the folded state to a greater extent in the test combination than in the control combination, the test ligand is a ligand that binds to the target protein; and (f) repeating steps (b)-(e) with a plurality of said test ligands until a ligand that binds to the target protein is identified.
Claim 6
Dependent Claim
23 words
The method of claim 5, wherein the ligand binds to surface or internal amino acid residues or conformational domains of the target protein.
Claim 7
Independent Claim
57 words
A method according to any of claims 1-6, wherein the determining step comprises subjecting the test and control combinations to at least one of proteolysis; antibody binding; surface binding; molecular chaperone binding; binding to a known ligand, cofactor, substrate, or analogue thereof of the target protein; circular dichroism spectroscopy; ultraviolet spectroscopy; fluorescence spectroscopy; calorimetry; and combinations thereof.
Claim 8
Dependent Claim
106 words
The method of Claim 7, wherein the determining step comprises the steps of: (i) incubating the target protein, each of said test ligands and one or more proteases which preferentially degrade target protein in its unfolded state, whereby the target protein in its unfolded state is degraded preferentially; and (ii) measuring the fraction of the target protein degraded or the fraction of the target protein remaining in an intact state, wherein if the fraction of target protein remaining in the intact state in the test combination is greater than in the control combination, then the test ligand is a ligand that binds to the target protein.
Claim 9
Dependent Claim
105 words
The method of Claim 7, wherein the determining step comprises the steps of: (i) combining the target protein and each of said test ligands; (ii) exposing the target protein and the test ligand mixture to a surface which preferentially binds unfolded target protein, whereby unfolded target protein binds to the surface; and (iii) determining the fraction of the target protein bound to the surface or the fraction of the target protein remaining unbound, wherein if the fraction of target protein remaining unbound is greater in the test combination, than in the control combination, the test ligand is a ligand that binds to the target protein.
Claim 10
Dependent Claim
94 words
The method of Claim 7, wherein the determining step comprises the steps of: (i) combining the target protein and each of said test ligands on a surface to which a known ligand of the target protein has been immobilized; and (ii) determining the fraction of the target protein bound to the surface or the fraction of the target protein remaining unbound wherein the fraction of target protein bound to the surface is greater in the test combination than in the control combination, the test ligand is a ligand that binds to the target protein.
Claim 11
Dependent Claim
26 words
The method of Claim 7, wherein the determining step comprises using antibodies that distinguish between the folded form and the unfolded form of the target protein.
Claim 12
Dependent Claim
64 words
The method of Claim 11, wherein the determining step comprises the steps of: (i) coating a surface with a specific antibody directed against the denatured state of the target protein; (ii) incubating the target protein in the presence of each of said test ligands on the surface; and (iii) determining the fraction of the target protein bound to or not bound to the surface.
Claim 13
Dependent Claim
86 words
The method of Claim 11, wherein the determining step comprises the steps of: (i) coating a surface with an antibody capable of binding the target protein in its native state or denatured state; (ii) incubating the target protein in the presence of each of said test ligands and an antibody capable of binding the target protein in its folded state or in the other of its native and denatured states; and (iii) determining the presence of the target protein bound or remaining unbound to the surface.
Claim 14
Dependent Claim
30 words
The method of Claim 7, wherein the determining step comprises determining the amount of target protein bound to a molecular chaperone protein or not bound to a molecular chaperone protein.
Claim 15
Dependent Claim
54 words
The method of Claim 14, wherein the determining step comprises the steps of: (i) coating a surface with a molecular chaperone protein; (ii) incubating the target protein and each of said test ligands on the coated surface; and (iii) determining the amount of the target protein bound to or remaining unbound to the surface.
Claim 16
Dependent Claim
77 words
The method of Claim 14, wherein the determining step comprises the steps of: (i) coating a surface with the target protein in a denatured state; (ii) incubating a purified form of the target protein in the presence of each of said test ligands and a molecular chaperone protein on the surface; and (iii) determining the amount of either or both of the molecular chaperone protein and the target protein remaining unbound to or binding to the surface.
Claim 17
Dependent Claim
48 words
The method of Claim 7, wherein the determining step comprises determining differential formation of aggregated protein using a method selected from the group consisting of: (i) measuring the amount of aggregated protein; (ii) measuring the amount of soluble protein and measuring the rate of formation of aggregated protein.
Claim 18
Dependent Claim
107 words
The method of Claim 7, wherein said binding to a known ligand, cofactor, substrate, or analogues thereof comprises the steps of: (i) immobilizing the known ligand, cofactor, substrate, or analogue thereof on a solid support; (ii) exposing the solid support to the test and control combinations, under conditions in which the target protein in its folded state binds to the support; and (iii) determining the fraction of the target protein bound to the support in the test and control combinations, the ligand being any test ligand that causes an increase or decrease in said fraction in the test combination relative to said fraction in the control combination.
Claim 19
Dependent Claim
25 words
The method of claim 8, wherein the proteases of the incubating step are selected from the group consisting of proteinase K, thermolysin, and combinations thereof.
Claim 20
Dependent Claim
21 words
The method of claim 14, wherein the determining step comprises subjecting the test and control combinations to denaturing polyacrylamide gel electrophoresis.
Claim 21
Dependent Claim
65 words
The method of Claim 7, wherein the determining step comprises the steps of: (i) coating a surface with antisera capable of binding to the folded target protein; (ii) incubating the target protein in the presence of a molecular chaperone and each of said test ligands on the surface; and (iii) determining the amount of the target protein binding to or remaining unbound to the surface.
Claim 22
Independent Claim
18 words
A method according to any of claims 1-21 wherein the target protein in the control combination unfolds irreversibly.
Claim 23
Independent Claim
18 words
A method according to any of claims 1-21 wherein the target protein in the control combination unfolds reversibly.
Claim 24
Independent Claim
32 words
A method according to any of claims 1-23 wherein the target protein is selected from the group consisting of carbonic anhydrase, human neutrophil elastase, human hemoglobin, dihydrofolate reductase, and HIV Rev protein.
References Cited (Backward Citations)
JPS4993093A
Cited
US4859609A
Cited
WO1992003542A1
Cited
US5506097A
Cited
WO1993014781A1
Cited
EP0613007A2
Cited
US5585277A
Cited
WO1997020952A1
Cited
US8082993B2
Cited
USPTO Annuity & Maintenance Windows
Maintenance schedule is calculated for granted US utility patents.