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US 2017/0306388 A1Published application
Method for the Identification of Inhibitors and Activators of Thymidylate Kinases
AssigneeCarnegie Mellon University
InventorsGordon Steven Rule, Kaustubh Sinha
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Abstract
Provided herein is a method of producing large amounts of the enzyme thymidylate kinase from multiple species for drug discovery purposes. Also provided herein are NMR methods of monitoring a reaction involving a thymidine kinase.
Claims16
A method of preparing an enzyme, comprising:
- a. culturing a cell comprising an exogenous gene for expressing an enzyme in cell culture medium under conditions suitable for expression of the exogenous gene in the cell;
- b. isolating the enzyme from the cell by lysing the cell in a lysis solution to produce a lysate, binding the enzyme in the lysate to a selectivity component specific to the enzyme that is optionally bound to a surface, and separating the bound enzyme from the lysate, wherein the enzyme is bound to the binding reagent in the presence of a substrate of the enzyme.
A method of monitoring an enzymatic reaction of a nucleoside phosphate kinase, comprising:
- a. conducting a reaction of the nucleoside phosphate kinase in a reaction mixture comprising a pyrimidine substrate of the nucleoside phosphate kinase having a pyrimidine H6 proton or a purine substrate of the nucleoside phosphate kinase having a purine H8 proton;
- and
- b. conducting a nuclear magnetic resonance (NMR) assay on the reaction mixture during the course of the reaction to identify or determine the presence of a shift in a peak of the spectrum corresponding to the pyrimidine H6 proton or purine H8 proton,
- wherein a shift in the peak corresponding to the pyrimidine H6 proton or purine H8 proton indicates enzymatic conversion of the substrate to the product of the enzymatic reaction.
1 more element
A method determining the effect of a composition on an enzymatic reaction of a nucleoside phosphate kinase, comprising:
- a. conducting a reaction of the nucleoside phosphate kinase in a reaction mixture comprising a pyrimidine substrate of the nucleoside phosphate kinase having a pyrimidine H6 proton or a purine substrate of the nucleoside phosphate kinase having a purine H8 proton and a sample composition;
- b. conducting a nuclear magnetic resonance (NMR) assay on the reaction mixture during the course of the reaction to identify or determine the presence of a shift in a peak of the spectrum corresponding to the pyrimidine H6 proton or the purine H8 proton, wherein a shift in the peak corresponding to the pyrimidine H6 proton or the purine H8 proton indicates enzymatic conversion of the substrate to the product of the enzymatic reaction;
- c. comparing, the rate and/or extent of enzymatic conversion of the substrate to the product of the enzymatic reaction to a rate and/or extent of enzymatic conversion of the substrate to the product of the enzymatic reaction in a reaction mixture either excluding the sample composition or having a different amount of the sample composition;
- and
- d. identifying sample compositions that affect the rate and/or extent of enzymatic conversion of the substrate to the product of the enzymatic reaction.
2 more elements
Dependencies are inferred from the claim text.