IN VITRO METHOD FOR PREDICTING THE EVOLUTIONARY RESPONSE OF HIV PROTEASE TO A DRUG TARGETED THEREAGAINST

摘要

An in vitro method for identifying distinct, first generation, drug-resistant, biologically active, HIV protease mutants that may emerge in vivo in response to a drug targeted thereagainst comprising: (a) preparing, in the presence of the drug, a library of all first-generation mutants of the protease differing therefrom by one to three amino acid substitutions, each of the protease mutants being generated as part of a polyprotein with reverse transcriptase; (b) isolating drug-resistant, biologically-active mutant proteases by assaying for activity of the reverse transcriptase; and (c) identifying the distinct amino acid changes leading to the drug-resistance of the active, mutant proteases so isolated. An in vitro method for evaluating the efficacy of a drug against a biologically active mutant or wild-type form of HIV protease comprising combining the drug and a mutant polyprotein, comprising an inactive HIV protease, HIV reverse transcriptase, and one or more protease cleavage sites, adding biologically-active mutant or wild-type protease, assaying for release of active reverse transcriptase, whereby reverse transcriptase activity indicates that the drug is not efficacious against the mutant or wild-type form of HIV protease tested.