Methods for identifying hot-spot residues of binding proteins and small compounds that bind to the same

Abstract

The present invention provides methods of identifying hot-spot residues for one or both members of a receptor-ligand complex of interest. Further provided are methods of using receptor hot-spot residues to identify compounds that functionally bind a receptor in a manner that mimics the binding of a known ligand for the receptor.

What is claimed is:  
     
         1 . A method for identifying a hot-spot residue of a member of a receptor-ligand complex comprising the steps of: 
 a. obtaining the protection factors of particular hydrogens on individual amino acid residues of which the member is composed; and    b. determining from the protection factors which individual amino acid residues of the member each contribute at least about 10% of the overall free energy of binding of the complex.    
     
     
         2 . The method of  claim 1  wherein said particular hydrogens are amide hydrogens on the peptide link between adjacent amino acid residues.  
     
     
         3 . The method according to  claim 2  wherein said individual amino acid residues comprise the binding surface of the member.  
     
     
         4 . The method according to  claim 2  wherein those hot-spot residues identified in sum contribute about 20-30% of the overall free energy of binding of the complex.  
     
     
         5 . The method according to  claim 2  wherein those hot-spot residues identified contribute about 40% of the overall free energy of binding of the complex.  
     
     
         6 . The method according to  claim 2 , in which the protection factors are obtained by a method comprising the steps of: 
 a. measuring the hydrogen exchange rates of the amide hydrogens of the individual amino acid residues composing the member when the member is in an unbound state;    b. measuring the hydrogen exchange rates of the amide hydrogens of the individual amino acid residues composing the member when the member is in a bound state; and    c. for each individual residue taking the ratio of the value obtained in step b with that of step a.    
     
     
         7 . The method according to  claim 6  wherein the exchange rates of the amide hydrogens of the individual residues of the member in the unbound state are determined by a method which comprises the steps of: 
 a. contacting the member with heavy hydrogen under conditions wherein amide hydrogens in the member which are accessible to solvent exchange with, and become labeled by, heavy hydrogen;  
 b. contacting the labeled receptor with a solvent that is substantially free of heavy hydrogen under rapid exchange conditions for varying “off-exchange” periods during which solvent-accessible amide hydrogens or heavy hydrogens of the labeled member exchange with normal hydrogen of the solvent;  
 c. for each “off-exchange” period, quantifying the amount of heavy hydrogen label associated with the amide hydrogen of each individual amino acid residue of the member; and  
 d. determining the hydrogen exchange rates for the amide hydrogens of each individual member residue from the quantity of label associated with that residue as a function of the varying “off-exchange” period.  
 
     
     
         8 . The method of  claim 7  wherein the amount of label associated with the amide hydrogens of each individual member residue for each off-exchange period is quantified by a method comprising the steps of: 
 a. fragmenting the labeled receptor into a plurality of fragments under conditions of slow hydrogen exchange;  
 b. determining which fragments are labeled with heavy hydrogen;  
 c. progressively degrading, under conditions of slow hydrogen exchange, each of the heavy hydrogen-labeled fragments to obtain a series of subfragments, wherein each subfragment of the series is composed of about 1-5 fewer amino acid residues than the preceding subfragment in the series;  
 d. quantifying the amount of heavy hydrogen associated with each subfragment; and  
 e. correlating the amount of heavy hydrogen of the subfragments with the amino acid sequences of the fragments from which the subfragments were generated, thereby localizing the positions of the fragment that had been labeled with heavy hydrogen to a resolution of about 1-5 amino acid residues.  
 
     
     
         9 . The method of  claim 8  in which the heavy hydrogen is tritium and the presence or amount of heavy hydrogen on a fragment or subfragment is determined by radioactivity measurements.  
     
     
         10 . The method of  claim 8  in which the heavy hydrogen is deuterium and the presence or amount of heavy hydrogen on a fragment or subfragment is determined by NMR spectroscopy.  
     
     
         11 . The method of  claim 8  further including the step of separating the fragments prior to determining which fragments are labeled with heavy hydrogen.  
     
     
         12 . The method of  claim 11  in which the separation comprises two sequential separations steps which are carried out under different conditions.  
     
     
         13 . The method of  claim 12  in which the first sequential separation step is carried out at a pH in the range of pH 2.1 to pH 3.0 and the second sequential separation step is carried out at a pH in the range of pH 2.1 to pH 3.0, where the pH of the first and second sequential separation steps are different.  
     
     
         14 . The method of  claim 13  in which the first sequential separation step is carried out at a pH of 2.7 and the second sequential separation step is carried out at a pH of 2.1.  
     
     
         15 . The method of  claim 8  in which the sequential degradation of the labeled fragments comprises contacting the labeled fragments with an acid resistant carboxypeptidase selected from the group consisting of carboxypeptidase P, carboxypeptidase Y, carboxypeptidase W and carboxypeptidase C.  
     
     
         16 . The method of  claim 8  in which the sequential degradation of the labeled fragments comprises contacting the labeled fragments with pentafluoropropionic acid anhydride.  
     
     
         17 . The method of  claim 8  in which any disulfide bridges in the off-exchanged member are disrupted under conditions of slow hydrogen exchange prior to fragmentation or progressive subfragmentation.  
     
     
         18 . The method of  claim 17  in which the disulfide bridges are disrupted by contacting the off-exchanged member or fragments with a water-soluble phosphine.  
     
     
         19 . The method of  claim 8  where prior to fragmentation or subfragmentation, the off-exchanged member is denatured under conditions of slow hydrogen exchange.  
     
     
         20 . The method of  claim 19  in which the denaturation is carried out in a solvent such that the pH for minimization of hydrogen exchange is substantially higher than that of a purely aqueous solution.  
     
     
         21 . The method of  claim 20  in which the solvent comprises about 5-20% water and the remainder is a nonaqueous polar solvent.  
     
     
         22 . The method of  claim 21  in which the nonaqueous polar solvent is selected from the group consisting of acetonitrile, dimethyl sulfoxide, a polyol and combinations thereof.  
     
     
         23 . The method of  claim 22  in which the polyol is glycerol.  
     
     
         24 . The method of  claim 21  in which the solvent further includes about 2-4 M guanidine thiocyanate.  
     
     
         25 . The method of  claim 21 , wherein the pH of the solvent is pH 4.8-5.2.  
     
     
         26 . The method of  claim 6  in which the amide hydrogen exchange rates of the individual residues of the member in the bound state are determined by a method which comprises the steps of: 
 a. contacting the member with a heavy hydrogen-labeled solvent under conditions of rapid hydrogen exchange for an “on-exchange” period sufficient for solvent-accessible amide hydrogens of the member to exchange with, and be substantially replaced by, heavy hydrogens of the solvent;  
 b. contacting the member with its binding partner under conditions wherein the heavy hydrogen labels are substantially retained and wherein the member binds its partner so as to form a receptor-ligand complex;  
 c. contacting the receptor-ligand complex with a solvent that is substantially free of heavy hydrogen under rapid exchange conditions for varying “off-exchange” periods during which solvent-accessible amide hydrogens or heavy hydrogens of the receptor-ligand complex exchange with normal hydrogen of the solvent;  
 d. for each “off-exchange” period, quantifying the amount of heavy hydrogen label associated with the amide hydrogens of individual amino acid residues of the receptor; and  
 e. determining the exchange rates of each individual member residue from the quantity of label associated with that residue as a function of the varying “off-exchange” periods.  
 
     
     
         27 . The method of  claim 26  wherein the amount of label associated with the amide hydrogens of the individual member residues for each off-exchange period is quantified by a method comprising the steps of: 
 a. dissociating the receptor-ligand complex and recovering the off-exchanged member of interest, both under conditions of slowed hydrogen exchange;  
 b. fragmenting the off-exchanged member into a plurality of fragments under conditions of slow hydrogen exchange;  
 c. determining which fragments are labeled with heavy hydrogen;  
 d. progressively degrading, under conditions of slow hydrogen exchange, each of the heavy hydrogen-labeled fragments to obtain a series of sub fragments, wherein each subfragment of the series is composed of about 1-5 fewer amino acid residues than the preceding subfragment in the series;  
 e. quantifying the amount of heavy hydrogen associated with each subfragment; and  
 f. correlating the amount of heavy hydrogen of the subfragments with the amino acid sequences of the fragments from which the subfragments were generated, thereby localizing the positions of the fragment that had been labeled with heavy hydrogen to a resolution about 1-5 amino acid residues.  
 
     
     
         28 . The method of  claim 27  in which the heavy hydrogen is tritium and the presence or amount of heavy hydrogen on a fragment or subfragment is determined by radioactivity measurements.  
     
     
         29 . The method of  claim 27  in which the heavy hydrogen is deuterium and the presence or amount of heavy hydrogen on a fragment or subfragment is determined by NMR spectroscopy or by measuring the mass of the fragment or subfragment.  
     
     
         30 . The method of  claim 27  further including the step of separating the fragments prior to determining which fragments are labeled with heavy hydrogen.  
     
     
         31 . The method of  claim 28  in which the separation comprises two sequential separations steps which are carried out under different conditions.  
     
     
         32 . The method of  claim 31  in which the first sequential separation step is carried out at a pH in the range of pH 2.1 to pH 3.0 and the second sequential separation step is carried out at a pH in the range of pH 2.1 to pH 3.0, where the pH of the first and second sequential separation steps are different.  
     
     
         33 . The method of  claim 32  in which the first sequential separation step is carried out at a pH of 2.7 and the second sequential separation step is carried out at a pH of 2.1.  
     
     
         34 . The method of  claim 27  in which the sequential degradation of the labeled fragments comprises contacting the labeled fragments with an acid resistant carboxypeptidase selected from the group consisting of carboxypeptidase P, carboxypeptidase Y, carboxypeptidase W and carboxypeptidase C.  
     
     
         35 . The method of  claim 27  in which the degradation of the labeled fragments comprises contacting the labeled fragments with pentafluoropropionic acid anhydride.  
     
     
         36 . The method of  claim 27  in which any disulfide bridges in the off-exchanged member are disrupted under conditions of slow hydrogen exchange prior to fragmentation or progressive subfragmentation.  
     
     
         37 . The method of  claim 36  in which the disulfide bridges are disrupted by contacting the off-exchanged member with a water-soluble phosphine.  
     
     
         38 . The method of  claim 27  where prior to fragmentation or subfragmentation, the off-exchanged member is denatured under conditions of slow hydrogen exchange.  
     
     
         39 . The method of  claim 38  in which the denaturation is carried out in a solvent such that the pH for minimization of hydrogen exchange is substantially higher than that of a purely aqueous solution.  
     
     
         40 . The method of  claim 39  in which the solvent comprises about 5-20% water and the remainder is a nonaqueous polar solvent.  
     
     
         41 . The method of  claim 40  in which the nonaqueous polar solvent is selected from the group consisting of acetonitrile, dimethyl sulfoxide, a polyol and combinations thereof.  
     
     
         42 . The method of  claim 41  in which the polyol is glycerol.  
     
     
         43 . The method of  claim 40  in which the solvent further includes about 2-4 M guanidine thiocyanate.  
     
     
         44 . The method of  claim 40 , wherein the pH of the solvent is pH 4.8-5.2.  
     
     
         45 . The method of  claim 6  wherein the exchange rates for the amide hydrogens of individual amino acid residues of the member in the unbound state are determined by deuterium or tritium exchange NMR.  
     
     
         46 . The method of  claim 6  wherein the exchange rates for the amide hydrogens of individual amino acid residues of the member in the bound state are determined by deuterium or tritium exchange NMR.  
     
     
         47 . The method of  claim 1  wherein said particular hydrogens are alkyl hydrogens on individual amino acid residues.  
     
     
         48 . The method according to  claim 47 , in which the protection factors are obtained by a method comprising the steps of: 
 a. measuring the hydrogen exchange rates of the alkyl hydrogens of the individual amino acid residues composing the member when the member is in an unbound state;    b. measuring the hydrogen exchange rates of the alkyl hydrogens of the individual amino acid residues composing the member when the member is in a bound state; and    c. for each individual residue taking the ratio of the value obtained in step b with that of step a.    
     
     
         49 . The method according to  claim 48  wherein the exchange rates of the alkyl hydrogens of the individual residues of the member in the unbound state are determined by a method which comprises the steps of: 
 a. contacting the member with heavy hydrogen under conditions wherein alkyl hydrogens in the member which are accessible to solvent exchange with, and become labeled by, heavy hydrogen for varying “on-exchange” periods;  
 b. for each “on-exchange” period in step a., quantifying the amount of heavy hydrogen label associated with the alkyl hydrogen of each individual amino acid residue of the member; and  
 c. determining the hydrogen exchange rates for the alkyl hydrogens of each individual member residue from the quantity of label associated with that residue as a function of the varying “on-exchange” period.  
 
     
     
         50 . The method of  claim 8  in which the presence or amount of heavy hydrogen on a fragment or subfragment is determined by measuring the mass of the fragment or subfragment by mass spectrometry.  
     
     
         51 . The method of  claim 8  in which the sequential degradation of the labeled fragments comprises application of collision induced mass spectrometry with a minimized scrambling threshold.  
     
     
         52 . The method of  claim 8  in which the sequential degradation of the labeled fragments comprises a combination of enzymatic fragmentation and collision induced mass spectrometry.  
     
     
         53 . A method of identifying a compound that binds a receptor at thermodynamically significant hot-spot residues bound by a known ligand for the receptor, comprising: 
 a. identifying the receptor hot-spot residues for the receptor-ligand pair of interest; and    b. selecting from a pool of candidate compounds those compounds that bind to at least one of the receptor hot-spot residues.    
     
     
         54 . The method of  claim 53  wherein the candidate compounds are screened for binding to the receptor and those compounds that bind are screened for binding to receptor hot-spot residues.  
     
     
         55 . A method of  claim 53  in which the receptor hot-spot residues are identified by the method of  claim 1 .  
     
     
         56 . The method of  claim 53  in which the receptor hot-spot residues are identified by the technique of site-directed mutagenesis.  
     
     
         57 . A method of identifying a compound which acts as an agonist for a receptor of interest, comprising: 
 a. identifying for a receptor-agonist pair of interest those receptor residues which participate in (i) receptor-agonist binding; (ii) receptor conformational changes as a consequence of the receptor-agonist binding event (class ii residues); and (iii) receptor subunit oligomerization (class iii residues); and    b. selecting from a pool of candidate compounds those compounds that interact with at least one class ii or class iii receptor residue.