Method for determining the binding behavior of ligands which specifically bind to target molecules

Abstract

The invention relates to a method for determining the binding behavior of ligands which specifically bind to target molecules at at least one binding site, whereby the markers are present in a native form and the determination of the concentrations K4 and K5 or of the quantities M2 and M1 is carried out by means of mass spectrometry.

1 . A species with a pharmaceutical activity, which pharmaceutical activity has been detected by a method of determining the ability of the species to bind specifically to one or more target molecules at at least one binding site thereof, the method comprising: 
 (A) preparing a first mixed phase wherein the species is contacted, alone or in combination with one or more other species, with (i) one or more markers which bind specifically to the one or more target molecules, and (ii) with the one or more target molecules, wherein the species is (are) present in a concentration K1, the one or more markers are present in a concentration K2 and the one or more target molecules are present in a concentration K3;    (B) preparing a second mixed phase wherein the one or more markers are contacted with the one or more target molecules;    (C) incubating the first and second mixed phases under identical conditions, which conditions permit a binding of the species and the one or more markers to the one or more target molecules, resulting in (a) a quantity M1 of one or more markers GM1 that are bound to one or more target molecules and a concentration K4 of one or more unbound markers in the first mixed phase and (b) a quantity M2 of one or more markers GM2 that are bound to one or more target molecules and a concentration K5 of one or more unbound markers in the second mixed phase;    (D) separating the one or more markers GM1 from the first mixed phase and separating the one or more markers GM2 from the second mixed phase;    (E1) determination of the concentrations K4 and K5, and calculating a concentration K6 from K5, wherein K6 is a concentration of one or more unbound markers in the second mixed phase under the assumption that the one or more markers have been contacted therein in the concentration K2 with the one or more target molecules in the concentration K3, and determining the binding behavior of the species from the ratio of the concentrations K6 and K4, or    (E2) determination of the quantities M1 and M2, and calculating a quantity M3 from M2, wherein M3 is a quantity of one or more bound markers in the second mixed phase under the assumption that the one or more markers have been contacted therein in the concentration K2 with the one or more target molecules in the concentration K3, and determining the binding behavior of the species from the ratio of the quantities M3 and M1;    wherein the one or more markers are present in a native form, the determination of K4 and K5 or M1 and M2 is carried out by mass spectrometry, and the species is a previously unknown species.    
   
   
       2 . The species of  claim 1 , wherein in (B) the one or more markers are present in a concentration K2 and the one or more target molecules are present in a concentration K3.  
   
   
       3 . The species of  claim 1 , wherein the one or more markers GM1 comprise specifically bound markers SGM1 and the one or more markers GM2 comprise specifically bound markers SGM2 and wherein SGM1 and SGM2 are released after (D), and quantities thereof are used in (E2) instead of M1 and M2.  
   
   
       4 . The species of  claim 1 , wherein the method further comprises repeating (A) to (E1) or (A) to (E2) wherein in at least one of (A) and (B) an agent which competes with the one or more markers for a specific binding to the one or more target molecules is added to at least one of the first and second mixed phases in an amount which substantially prevents or cancels a specific binding of the one or more markers to the one or more target molecules.  
   
   
       5 . The species  claim 1 , wherein the method further comprises repeating (A) to (E1) or (A) to (E2) at least once with a different ratio of K1 and K3.  
   
   
       6 . The species of  claim 1 , wherein (D) comprises at least one of dialysis, precipitation, adsorption, binding to immobilized molecules which have an affinity to at least one of the one or more target molecules, complexes of the one or more target molecules and the one or more markers and complexes of the one or more target molecules and the one or more species, centrifugation, chromatography, and filtration.  
   
   
       7 . The species of  claim 1 , wherein (D) comprises an ultrafiltration.  
   
   
       8 . The species of  claim 1  , wherein (D) is carried out under conditions under which at least one of (a) the concentrations of the one or more unbound markers in the first and the second mixed phases and (b) the amounts of the one or more bound markers GM1 and GM2 remain constant.  
   
   
       9 . The species of  claim 8 , wherein the mass spectrometry is carried out in one of the MS, MS/MS and MS n  modes.  
   
   
       10 . The species of  claim 9 , wherein the mass spectrometry is preceded by at least one of a capillary electrophoresis, a gas chromatography and a liquid chromatography.  
   
   
       11 . The species of  claim 1 , wherein the mass spectrometry is preceded by a HPLC.  
   
   
       12 . The species of  claim 11 , wherein the HPLC comprises reverse phase chromatography.  
   
   
       13 . The species of  claim 1 , wherein prior to the mass spectrometry a sample preparation by means of at least one of extraction, filtration, gas chromatography, capillary electrophoresis, liquid chromatography, FPLC®, reverse phase chromatography, size exclusion chromatography and affinity chromatography is carried out.  
   
   
       14 . The species of  claim 1 , wherein the species has been generated by a method of combinatorial chemistry.  
   
   
       15 . The species of  claim 1 , wherein the one or more target molecules comprise at least one target molecule that is embedded in a membrane structure.  
   
   
       16 . The species of  claim 1 , wherein at least one of the one or more target molecules and the one or more markers is selected from peptides, proteins, hormones, nucleic acids, sugars, polymers, low molecular weight compounds of natural or synthetic origin and structures on or inside cells, cell fragments, cell homogenates, synaptosomes, liposomes, vesicles, tissue cuts, viruses, components and fragments thereof, capsides, components and fragments thereof, and mixtures of natural substances.  
   
   
       17 . The species of  claim 1 , wherein at least one of the one or more target molecules and the one or more markers is selected from receptors, prions, enzymes, transport proteins, ion channels, antibodies, synaptic plasma membrane vesicles and vesicles found inside cells.  
   
   
       18 . The species of  claim 1 , wherein the species is selected from peptides, proteins, hormones, nucleic acids, sugars, polymers, low molecular weight compounds of natural or synthetic origin and structures on or inside cells, cell fragments, cell homogenates, synaptosomes, liposomes, vesicles, tissue cuts, viruses, components and fragments thereof, capsides, components and fragments thereof, and mixtures of natural substances.  
   
   
       19 . The species of  claim 1 , wherein the species is selected from receptors, prions, enzymes, transport proteins, ion channels, antibodies, synaptic plasma membrane vesicles and vesicles found inside cells.  
   
   
       20 . A pharmaceutical composition which comprises the species of  claim 1  and a pharmaceutically acceptable carrier.