Method for high throughput thermodynamic screening of ligands

Abstract

The present invention provides a method of rapidly screening ligands on the basis of their binding affinity and binding enthalpy by carrying out binding assays at a minimum of two temperatures. This technique permits the selection for further optimization of lead ligands that bind to the target molecules with favorable enthalpies. Those ligands will exhibit higher solubilities in aqueous solution than ligands selected by conventional means, and may exhibit lower susceptibilities to resistant mutations. The method may be utilized as a standalone technique, or may be adapted to, for example, known high throughput screening technologies. The present invention further comprises an apparatus for carrying out the described methods.

We claim:  
     
         1 . A method for screening samples on the basis of binding enthalpy, comprising the steps of: 
 a) exposing a macromolecule to a plurality of candidate ligands at a minimum of two different temperatures;    b) exposing an identical macromolecule to said plurality of candidate ligands at said minimum of two different temperatures;    c) measuring a binding affinity for each of said plurality of candidate ligands at each of said minimum of two different temperatures, producing a plurality of binding affinities; and    d) comparing said plurality of binding affinities.    
     
     
         2 . The method of    claim 1    further comprising the step of selecting lead ligands based on said comparison of said plurality of binding affinities.  
     
     
         3 . The method of    claim 1    wherein said macromolecule is selected from the group consisting of: proteins, peptides, nucleic acids, carbohydrates, and receptor molecules.  
     
     
         4 . The method of    claim 1    wherein said candidate ligands are selected from the group consisting of: proteins, peptides, nucleic acids, carbohydrates, enzyme substrates, and enzyme inhibitors.  
     
     
         5 . The method of    claim 1    wherein said vessel is selected from the group consisting of: multi-well plates, capillary tubes, etched channels, solid supports, and cuvettes.  
     
     
         6 . The method of    claim 1    wherein said macromolecule is immobilized in said vessel.  
     
     
         7 . The method of    claim 1    wherein said step of measuring is carried out by detecting changes in a parameter selected from the group consisting of: 
 fluorescence, luminescence, emission, absorption, radioactivity, fluorescence resonance energy transfer, pH, enzymatic activity, molecular weight, and antibody binding.  
 
     
     
         8 . An apparatus for screening samples on the basis of binding enthalpy, comprising: 
 a) means for exposing a macromolecule to a plurality of candidate ligands at a first temperature; and    b) means for exposing an identical macromolecule to said plurality of candidate ligands at a minimum of one different temperature.    
     
     
         9 . The apparatus of    claim 8   , further comprising means for measuring a binding affinity for each of said plurality of candidate ligands at said first temperature and at said minimum of one different temperature.  
     
     
         10 . The apparatus of    claim 9   , further comprising means for storing and displaying said measured binding affinities.  
     
     
         11 . The apparatus of    claim 9   , further comprising means for analyzing said measured binding affinities.