In vitro method for predicting the evolutionary response of a protein to a drug targeted thereagainst

Abstract

Inhibitory drug combinations targeted against a protein and first-generation, drug- resistant mutants of the protein. In a preferred embodiment, a combination of drugs that inhibits the activity of a protein and all first-generation, drug-resistant mutants of the protein is identified by determining in vitro the identity of each distinct, first-generation, drug-resistant mutant form of a protein that may arise in vivo in response to a first drug targeted thereagainst, wherein the distinct, first-generation, drug-resistant mutants contain a limited number of resistance-conferring mutations, and then determining in vitro the identity of one or more auxiliary drugs that inhibit all of the first-generation, drug-resistant mutants of the protein, wherein the first drug and the one or more auxiliary drugs represent the combination of drugs.

What is claimed is:  
     
         1 . A method for predicting, in vitro, distinct, drug-resistant, biologically- active mutants of a protein that may emerge in vivo in response to a drug targeted thereagainst, said method comprising the steps of: 
 (a) providing a library of nucleotide sequences, said nucleotide sequences encoding mutant proteins that differ from the original protein by, at least one amino acid substitution;    (b) expressing said library of nucleotide sequences by heterologous expression to provide a library of mutant proteins;    (c) isolating, in vitro, drug-resistant, biologically-active mutant proteins from said library of mutant proteins; and    (d) identifying the mutant proteins so isolated, whereby every mutant protein so identified for which another mutant so isolated having the same amino acid sequence is not also identified represents a distinct, drug-resistant, biologically-active mutant that may emerge in vivo in response to the drug.    
     
     
         2 . The method as claimed in    claim 1   , wherein said nucleotide sequences encode mutant proteins that differ from the original protein by at least one and not more than three amino acid substitutions.  
     
     
         3 . The method as claimed in    claim 1    wherein said nucleotide sequences encode mutant proteins that differ from the original protein by at least one and not more than two amino acid substitutions.  
     
     
         4 . The method as claimed in    claim 1    wherein said nucleotide sequences encode mutant proteins that differ from the original protein by a single amino acid substitution.  
     
     
         5 . The method as claimed in    claim 1    wherein said step of providing a library of nucleotide sequences comprises synthesizing a randomized library of isolated nucleotide sequences encoding mutant proteins that differ from the original protein by at least one amino acid substitution.  
     
     
         6 . The method as claimed in    claim 1    wherein said step of providing a library of nucleotide sequences comprises synthesizing a defined library of isolated nucleotide sequences encoding mutant proteins that differ from the original protein by at least one amino acid substitution.  
     
     
         7 . A method for predicting, in vitro, distinct, drug-resistant, biologically-active mutants of a protein that may emerge in vivo in response to a drug targeted thereagainst, said method comprising the steps of: 
 (a) synthesizing a library of isolated nucleotide sequences, said library of isolated nucleotide sequences encoding mutant proteins that differ from the original protein by at least one amino acid substitution;    (b) expressing said library of isolated nucleotide sequences to provide a library of mutant proteins;    (c) isolating, in vitro, drug-resistant, biologically-active mutant proteins from said library of mutant proteins; and    (d) identifying the mutant proteins so isolated, whereby every mutant protein so identified for which another mutant so isolated having the same amino acid sequence is not also identified represents a distinct, drug-resistant, biologically-active mutant that may emerge in vivo in response to the drug.    
     
     
         8 . The method as claimed in    claim 7    wherein said isolated nucleotide sequences encode mutant proteins that differ from the original protein by at least one and not more than three amino acid substitutions.  
     
     
         9 . The method as claimed in    claim 7    wherein said isolated nucleotide sequences encode mutant proteins that differ from the original protein by at least one ,d not more than two amino acid substitutions.  
     
     
         10 . The method as claimed in    claim 7    wherein said isolated nucleotide sequences encode mutant proteins that differ from the original protein by a single amino acid substitution.  
     
     
         11 . A method of predicting, in vitro, each distinct, first-generation, drug-resistant, biologically-active mutant of a protein that may merge in vivo in response to a drug targeted thereagainst, the method comprising the steps of: 
 (a) producing a library of mutants of the protein, said library including every protein that differs from the original protein or a region thereof by at least one amino acid substitution;    (b) isolating, in vitro, each drug-resistant, biologically-active, mutant protein from said library; and    (c) identifying each mutant protein so isolated, whereby every mutant protein so identified for which another mutant so isolated having the same amino acid sequence is not also identified represent a distinct first-generation, drug-resistant, biologically-active mutant that may emerge in vivo in response to the drug.    
     
     
         12 . The method as claimed in    claim 11    wherein said library of mutants includes every protein that differs from the original protein or a region thereof by at least one and not more than three amino acid substitutions.  
     
     
         13 . The method as claimed in    claim 11    wherein said library of mutants includes every protein that differs from the original protein or a region thereof by at least one and not more than two amino acid substitutions.  
     
     
         14 . The method as claimed in    claim 11    wherein said library of mutants includes every protein that differs from the original protein or a region thereof by a single amino acid substitution.  
     
     
         15 . The method as claimed in    claim 11    wherein said library of mutants is produced by synthesizing a randomized library of mutant nucleotide sequences encoding said mutant proteins, said mutant nucleotide sequences of said randomized library encoding an average of up to three amino acid substitutions per variant protein molecule, and then expressing said mutant nucleotide sequences.  
     
     
         16 . The method as claimed in    claim 11    wherein said library of mutants is produced by synthesizing a defined library of mutant nucleotide sequences encoding said mutant proteins, said mutant nucleotide sequences of said defined library encoding up to three amino acid substitutions per variant protein molecule, and then expressing said mutant nucleotide sequences.  
     
     
         17 . The method as claimed in    claim 11    wherein the protein is the HIV-1 protease.  
     
     
         18 . The method as claimed in    claim 17    wherein said mutants of step (a) are expressed as part of the HIV-1 polyprotein and wherein said isolating step (b) comprises selecting for autocatalysis of the HIV-1 polyprotein.  
     
     
         19 . The method as claimed in    claim 18    wherein the HIV-1 polyprotein is expressed as a fusion protein on the surface of a phage.  
     
     
         20 . The method as claimed in    claim 18    wherein the HIV-1 polyprotein is expressed as a fusion protein in a two hybrid system.  
     
     
         21 . The method as claimed in    claim 18    wherein the HIV-1 polyprotein is expressed as a fusion protein with an affinity ligand.  
     
     
         22 . The method as claimed in    claim 21    wherein said affinity ligand is selected from the group consisting of maltose binding protein, FLAG antigen, biotinylated peptide, polyhistidine and β-galactosidase.  
     
     
         23 . A method of evaluating the ultimate clinical efficacy of a first drug which inhibits the activity of a protein, the method comprising the steps of: 
 (a) determining, in vitro, the number of distinct, first-generation, biologically-active mutants of the protein displaying resistance to said first drug; and    (b) comparing said number to standards obtained from other drugs whose relative in vivo efficacies are known;    whereby said first drug may be predicted to have a relatively greater ultimate clinical efficacy than said other drugs if the number of distinct, first-generation, biologically-active mutants displaying resistance to said first drug is smaller than the number of distinct, first-generation, biologically-active mutants displaying resistance to said other drugs.    
     
     
         24 . The method as claimed in    claim 23    wherein the in vitro determination of the number of distinct, first-generation, biologically-active, drug-resistant mutants comprises the steps of 
 (a) producing a library of mutants of the protein, said library including every protein that differs from the original protein or a region thereof by at least one amino acid substitution;  
 (b) isolating, in vitro, each drug-resistant, biologically-active, mutant protein from said library;  
 (c) identifying each mutant protein so isolated, whereby every mutant protein so identified for which another mutant so isolated having the same amino acid sequence is not also identified represents a distinct, first-generation, drug-resistant, biologically-active mutant; and  
 (d) counting the number of distinct, first-generation, drug-resistant, biologically-active mutants thus identified.  
 
     
     
         25 . The method as claimed in    claim 24    wherein said library includes every protein that differs from the original protein or a region thereof by at least one and not more than three amino acid substitutions.  
     
     
         26 . The method as claimed in    claim 24    wherein said library includes every protein that differs from the original protein or a region thereof by at least one and not more than two amino acid substitutions.  
     
     
         27 . The method as claimed in    claim 24    wherein said protein is an HIV-1 protein.  
     
     
         28 . The method as claimed in    claim 27    wherein said protein is HIV-1 protease.  
     
     
         29 . A method of comparing, a priori, the relative ultimate clinical efficacies of two or more different drugs targeted against a single protein, the method comprising the steps of: 
 (a) determining, in vitro, under substantially identical conditions, the respective numbers of distinct, first-generation, biologically-active mutants which display resistance to each of the respective drugs; and    (b) comparing the respective numbers, whereby the drug which elicits the smallest number of such mutants is determined to have the greatest ultimate clinical efficacy.    
     
     
         30 . The method as claimed in    claim 29    wherein the in vitro determination of the number of distinct, first-generation, biologically-active, drug-resistant mutants for each of the respective drugs comprises the steps of: 
 (a) producing a library of mutants of the protein, said library including every protein that differs from the original protein or a region thereof by at least one amino acid substitution;  
 (b) isolating, in vitro, each drug-resistant, biologically-active, mutant protein from said library;  
 (c) identifying each mutant protein so isolated, whereby every mutant protein so identified for which another mutant so isolated having the same amino acid sequence is not also identified represents a distinct, first-generation, drug-resistant, biologically-active mutant; and  
 (d) counting the number of distinct, first-generation, drug-resistant, biologically-active mutants thus identified.  
 
     
     
         31 . A method of identifying a combination of drugs effective against a protein without the development by the protein of drug resistance, said method comprising the steps of: 
 (a) determining in vitro the identity of each distinct, first-generation, drug-resistant, biologically active mutant form of the protein that may arise in vivo in response to a first drug, wherein said distinct, first-generation, drug-resistant, biologically-active mutant forms contain a limited number of resistance-conferring mutations; and    (b) determining in vitro the identity of one or more auxiliary drugs that are effective against all of regeneration, drug-resistant, biologically active, mutant forms of the protein, wherein the combination of said first drug and said one or more auxiliary drugs constitutes said effective combination of drugs.    
     
     
         32 . The combination of drugs identified by the method of    claim 31   .  
     
     
         33 . A method of identifying a combination of drugs for use against a protein, said method comprising the steps of: 
 (a) determining in vitro the identity of a resistance-conferring mutation of the protein that may arise in response to a first drug; and    (b) determining in vitro the identity of an auxiliary drug which is effective against a mutant protein containing said resistance-conferring mutation and which interacts with said mutant protein at the site said resistance-conferring mutation, wherein the combination of said first drug and said one or ore auxiliary drugs constitutes said combination of drugs.    
     
     
         34 . The method as claimed in    claim 33    further comprising the steps of (a) and (b) for every additional resistance-conferring mutation.  
     
     
         35 . The combination of drugs identified by the method of    claim 33   .  
     
     
         36 . A method of identifying a drug effective against a first-generation, biologically-active mutant of an original protein, said method comprising the steps of: 
 (a) producing a library of first-generation mutants of the protein, said library including every protein that differs from the original protein or a region thereof by at least one amino acid substitution;    (b) determining which of said mutants possess biological activity; and    (c) testing prospective drugs in vitro against the biologically-active mutants in said library until a drug is identified which is effective against a first-generation, biologically-active mutant.    
     
     
         37 . The drug identified by the method of    claim 36   .  
     
     
         38 . A method of identifying a randomized peptide or nucleotide effective against a first-generation, biologically-active mutant of an original protein, said method comprising the steps of: 
 (a) producing a library of first-generation mutants of the protein, said library including every protein that differs from the original protein or a region thereof by at least one amino acid substitution;    (b) determining which of said mutants possess biological activity;    (c) generating a randomize peptide or nucleotide;    (d) testing the efficacy of said randomized peptide or nucleotide in vitro against the biologically-active mutants in said library; and    (e) repeating steps (c) and (d) until a randomized peptide or nucleotide is identified which is effective against a first-generation, biologically-active mutant.    
     
     
         39 . The randomized peptide or nucleotide identified by the method of    claim 38   .