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

A method for identifying, in vitro, distinct, drug-resistant, biologically-active mutants of an original viral protease that may emerge in vivo in response to a drug targeted against said protease, said method comprising the steps of:

• (a) synthesizing a library of isolated nucleotide sequences, said library of isolated nucleotide sequences encoding mutant polyproteins, Each comprising a mutant viral protease that differs from the original viral protease by at least one amino acid substitution;
• (b) causing the heterologous expression of said library of isolated nucleotide sequences to provide a library of mutant polyproteins that include the mutant protease and a native reverse transcriptase;
• (c) isolating, in vitro, mutant polyproteins that include drug-resistant proteases from said library of mutant polyproteins;
• and
• (d) identifying the mutant proteases in said polyproteins so isolated, whereby every mutant protease 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 identifying, in vitro, each distinct, first-generation, drug-resistant, biologically-active mutant of a protein that may emerge in vivo in response to a drug targeted against said protease, 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 between one and three amino acid substitutions, wherein said library of mutants of the protein are produced by generating a library of mutant nucleotide sequences encoding said mutant proteins and then causing the heterologous expression of said mutant nucleotide sequences;
• (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 represents 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 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 vivo in response to a first drug;
• (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 of said resistance-conferring mutation, wherein the combination of said first drug and said one or more auxiliary drugs constitutes said combination of drugs;
• and
• (c) repeating steps (a) and (b) for every additional resistance-conferring mutation.

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 between one and three amino acid substitutions, wherein said library of first-generation mutants of the protein are produced by generating a library of mutant nucleotide sequences encoding said mutant proteins and then causing the expression of said mutant nucleotide sequences;
• (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 randomly generated 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) randomly generating a peptide or nucleotide;
• (d) testing the efficacy of said randomly generated peptide or nucleotide in vitro against the biologically-active mutants in said library;
• and
• (e) repeating steps (c) and (d) until a randomly generated peptide or nucleotide is identified which is effective against a first-generation, biologically-active mutant.

A method for identifying, in vitro, distinct, first-generation, drug-resistant, biologically-active mutants of an original protein that may emerge in vivo in response to a drug targeted against said original protein, said method comprising the steps of:

• (a) causing the heterologous expression of nucleotide sequences, wherein said nucleotide sequences encode a comprehensive library of first-generation mutants of the original protein, to provide said comprehensive library of first-generation mutants of the original protein;
• (b) isolating, in vitro, drug-resistant, biologically-active mutant proteins from said library of first-generation mutants of the original protein;
• and
• (c) 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 first-generation mutant that may emerge in vivo in response to the drug.