Compositions and methods for monitoring the phosphorylation of natural binding partners
AssigneeCyclacel Limited
InventorsRoger K. Craig, John Colyer
Life of this patent
Estimating the term…
Abstract
This invention relates to methods and compositions for monitoring the interaction of binding partners as a function of the addition or subtraction of a phosphate group to or from one of the binding partners by a protein kinase or phosphatase.
Claims49
An isolated natural binding domain and a binding partner therefor, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner dependent upon phosphorylation or dephosphorylation of said site.
A kit comprising an isolated natural binding domain and a binding partner therefor, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner dependent upon phosphorylation of said site, and packaging material therefor.
A method for monitoring activity of an enzyme comprising performing a detection step to detect binding of an isolated natural binding domain and a binding partner therefor as a result of contacting one or both of said isolated natural binding domain and said binding partner with said enzyme, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner dependent upon phosphorylation of said site and wherein detection of binding of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of enzyme activity.
A method for monitoring activity of an enzyme comprising performing a detection step to detect dissociation of an isolated natural binding domain from a binding partner therefor as a result of contacting one or both of said isolated natural binding domain and said binding partner with said enzyme, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner dependent upon phosphorylation of said site and wherein detection of dissociation of said isolated natural binding domain from said binding partner as a result of said contacting is indicative of enzyme activity.
A method for monitoring the activity of a modulator of the activity of an enzyme comprising:
- a) mixing an isolated natural binding domain, a binding partner of said isolated natural binding domain, said enzyme, and a candidate modulator which binds to said isolated natural binding domain, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner dependent upon phosphorylation of said site, and wherein the combination of said isolated natural binding domain and said binding partner comprises detection means for monitoring association or dissociation of said isolated natural binding domain and said binding partner, and wherein detection of binding of said isolated natural binding domain and said binding partner as a result of said mixing is indicative of enzyme activity, and wherein said phosphorylation of said site occurs prior to said mixing step;
- and
- b) monitoring association or dissociation of said isolated natural binding domain and said binding partner, said association or dissociation being indicative of modulation by said candidate modulator of said activity, wherein said modulator reduces binding of said isolated natural binding domain and said binding partner and wherein a reduction in binding is detected by said detection means.
A method for monitoring the activity of a modulator of the activity of an enzyme comprising:
- a) mixing an isolated natural binding domain, a binding partner of said isolated natural binding domain, said enzyme, and a candidate modulator which binds to said isolated natural binding domain, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner dependent upon phosphorylation of said site, and wherein the combination of said isolated natural binding domain and said binding partner comprises detection means for monitoring association or dissociation said isolated natural binding domain and said binding partner, and wherein detection of binding of said isolated natural binding domain and said binding partner as a result of said mixing is indicative of enzyme activity, and wherein said phosphorylation of said site occurs during said mixing step;
- and
- b) monitoring association or dissociation of said isolated natural binding domain and said binding partner, said association or dissociation being indicative of modulation by said candidate modulator of said activity, wherein said modulator reduces binding of said isolated natural binding domain and said binding partner and wherein a reduction in binding is detected by said detection means.
A method for monitoring the activity of a modulator of the activity of an enzyme comprising:
- a) mixing an isolated natural binding domain, a binding partner of said isolated natural binding domain, said enzyme, and a candidate modulator which binds to said binding partner, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner dependent upon phosphorylation of said site, and wherein the combination of said isolated natural binding domain and said binding partner comprises detection means for monitoring association or dissociation of said isolated natural binding domain and said binding partner, and wherein detection of binding of said isolated natural binding domain and said binding partner as a result of said mixing is indicative of enzyme activity, and wherein said phosphorylation of said site occurs prior to said mixing step;
- and
- b) monitoring association or dissociation of said isolated natural binding domain and said binding partner, said association or dissociation being indicative of modulation by said candidate modulator of said activity, wherein said modulator reduces binding of said isolated natural binding domain and said binding partner and wherein a reduction in binding is detected by said detection means.
A method for monitoring the activity of a modulator of the activity of an enzyme comprising:
- a) mixing an isolated natural binding domain, a binding partner of said isolated natural binding domain, said enzyme, and a candidate modulator which binds to said binding partner, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner dependent upon phosphorylation of said site, and wherein the combination of said isolated natural binding domain and said binding partner comprises detection means for monitoring association or dissociation of said isolated natural binding domain and said binding partner, and wherein detection of binding of said isolated natural binding domain and said binding partner as a result of said mixing is indicative of enzyme activity, and wherein said phosphorylation of said site occurs during said mixing step;
- and
- b) monitoring association or dissociation of said isolated natural binding domain and said binding partner, said association or dissociation being indicative of modulation by said candidate modulator of said activity, wherein said modulator reduces binding of said isolated natural binding domain and said binding partner and wherein a reduction in binding is detected by said detection means.
A method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising
- a) mixing an isolated natural binding domain, a binding partner therefor and an enzyme with a candidate modulator of said kinase or phosphatase which binds said isolated natural binding domain, wherein said natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of said site by said kinase or phosphatase and wherein the combination of said isolated natural binding domain and said binding partner comprises a detection means for monitoring association or dissociation between said isolated natural binding domain and said binding partner, and wherein said phosphorylation or dephosphorylation occurs prior to said mixing, and
- b) monitoring the association or dissociation of said isolated natural binding domain to said binding partner, wherein association or dissociation of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of modulation of enzymatic activity by said candidate modulator of said kinase or phosphatase.
A method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising
- a) mixing an isolated natural binding domain, a binding partner therefor and an enzyme with a candidate modulator of said kinase or phosphatase which binds to said isolated natural binding domain, wherein said natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of said site by said kinase or phosphatase and wherein the combination of said isolated natural binding domain and said binding partner comprises a detection means for monitoring association or dissociation between said isolated natural binding domain and said binding partner, and wherein said phosphorylation or dephosphorylation occurs during said mixing, and
- b) monitoring the association or dissociation of said isolated natural binding domain to said binding partner, wherein association or dissociation of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of modulation of enzymatic activity by said candidate modulator of said kinase or phosphatase.
A method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising
- a) mixing an isolated natural binding domain, a binding partner therefor and an enzyme with a candidate modulator of said kinase or phosphatase which binds said binding partner, wherein said natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of said site by said kinase or phosphatase and wherein the combination of said isolated natural binding domain and said binding partner comprises a detection means for monitoring association or dissociation between said isolated natural binding domain and said binding partner, and wherein said phosphorylation or dephosphorylation occurs prior to mixing, and
- b) monitoring the binding of said isolated natural binding domain to said binding partner, wherein binding or dissociation of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of modulation of enzymatic activity by said candidate modulator of said kinase or phosphatase.
A method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising
- a) mixing an isolated natural binding domain, a binding partner therefor and an enzyme with a candidate modulator of said kinase or phosphatase which binds said binding partner, wherein said natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of said site by said kinase or phosphatase and wherein the combination of said isolated natural binding domain and said binding partner comprises a detection means for monitoring association or dissociation between said isolated natural binding domain and said binding partner, and wherein said phosphorylation or dephosphorylation occurs during said mixing, and
- b) monitoring the association or dissociation of said isolated natural binding domain to said binding partner, wherein association or dissociation of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of modulation of enzymatic activity by said candidate modulator of said kinase or phosphatase.
A method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising
- a) mixing an assay system comprising an isolated natural binding domain and a binding partner for said isolated natural binding partner with a candidate modulator of enzymatic activity of a said kinase or phosphatase which binds to said isolated natural binding domain, and
- b) monitoring association or dissociation of an isolated natural binding domain and a binding partner therefor in said assay system, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of said site by a said kinase or phosphatase in said assay system, wherein the combination of said isolated natural binding domain and said binding partner comprises a detection means for monitoring association or dissociation between said isolated natural binding domain and said binding partner and wherein said phosphorylation or dephosphorylation occurs prior to said mixing, and wherein association or dissociation of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of modulation of enzymatic activity by said candidate modulator of a said kinase or phosphatase.
A method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising
- a) mixing an assay system comprising an isolated natural binding domain and a binding partner for said isolated natural binding domain with a candidate modulator of enzymatic activity of a said kinase or phosphatase which binds to said isolated natural binding domain, and
- b) monitoring association or dissociation of an isolated natural binding domain and a binding partner therefor in said assay system, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of said site by a said kinase or phosphatase in said assay system, wherein the combination of said isolated natural binding domain and said binding partner comprises a detection means for monitoring association or dissociation between said isolated natural binding domain and said binding partner and wherein said phosphorylation or dephosphorylation occurs during said mixing, and wherein association or dissociation of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of modulation of enzymatic activity by said candidate modulator of a said kinase or phosphatase.
A method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising
- a) mixing an assay system comprising an isolated natural binding domain and a binding partner with a candidate modulator of enzymatic activity of a said kinase or phosphatase which binds to said binding partner, and
- b) monitoring association or dissociation of an isolated natural binding domain and a binding partner therefor in said assay system, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of said site by a said kinase or phosphatase in said assay system, wherein the combination of said isolated natural binding domain and said binding partner comprises a detection means for monitoring association or dissociation between said isolated natural binding domain and said binding partner and wherein said phosphorylation or dephosphorylation occurs prior to said mixing, and wherein association or dissociation of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of modulation of enzymatic activity by said candidate modulator of a said kinase or phosphatase.
A method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising
- a) mixing an assay system comprising an isolated natural binding domain and a binding partner with a candidate modulator of enzymatic activity of a said kinase or phosphatase which binds to said binding partner, and
- b) monitoring binding of an isolated natural binding domain and a binding partner therefor in said assay system, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of said site by a said kinase or phosphatase in said assay system, wherein the combination of said isolated natural binding domain and said binding partner comprises a detection means for monitoring association or dissociation between said isolated natural binding domain and said binding partner and wherein said phosphorylation or dephosphorylation occurs during said mixing, and wherein association or dissociation of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of modulation of enzymatic activity by said candidate modulator of a said kinase or phosphatase.
Dependencies are inferred from the claim text.