Sepracor, Inc.

Laurence M Melnick

Donald L Heefner

An in vitro method for predicting the identity of distinct, first-generation, drug-resistant, biologically-active, HIV protease mutants that may emerge in vivo in response to a drug targeted thereagainst. In a preferred embodiment, the in vitro method comprises the steps of (a) preparing, in the presence of the drug, a comprehensive library of all first-generation mutants of the protease differing therefrom by at least one and preferably no more than three amino acid substitutions, each of the protease mutants being generated as part of a polyprotein with the HIV reverse transcriptase protein; (b) isolating, in vitro, first-generation, drug-resistant, biologically-active, mutant proteases from said library by assaying for biological activity of the reverse transcriptase protein; and (c) identifying the distinct, first-generation, biologically-active, mutant proteases so isolated. The present invention also relates to an in vitro method for evaluating the efficacy of a drug against a biologically-active mutant or wild-type form of HIV protease, said method comprising the steps of (a) providing a mutant polyprotein, said mutant polyprotein including a biologically-inactive mutant form of the protease linked to HIV reverse transcriptase by one or more sites cleavable by the biologically-active or wild-type form of the protease; (b) adding the drug to the mutant polyprotein; (c) then, adding the biologically-active or wild-type form of the protease to the mutant polyprotein; and (d) then, assaying for the presence of biological activity for reverse transcriptase, whereby the presence of reverse transcriptase activity indicates that the drug is not efficacious against the biologically-active mutant or wild-type form of the protease tested. The present invention further relates to a kit for evaluating the efficacy of a drug against a biologically-active mutant or wild-type form of HIV protease.

In vitro method for predicting the evolutionary response of HIV protease

A method for identifying, in vitro, distinct, drug-resistant, biologically-active mutants of a protein that may emerge in vivo in response to a drug targeted against the protein is disclosed. The method involves preparing, by heterologous expression of a library of nucleotide sequences, a complete library of mutant proteins accessible in a single generation. The mutant proteins from the library that are drug resistant are identified. When all the first generation mutants have been identified in the above manner, a combination of drugs can be identified that will block the development of resistance. The same technique allows evaluation of the ultimate clinical efficacy of a drug targeted against the protein by comparing the number of resistant first generation mutants. A preferred protein is an HIV protease.

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Watertown, MA, United States of America

Laurence M Melnick