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

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.

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.

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.

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.

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.