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AssigneeSepracor Inc.
InventorsLaurence M. Melnick, Donald L. Heefner
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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.
A kit for evaluating, in vitro, the efficacy of a drug against a biologically-active mutant or wild-type form of a first protein, the first protein being a protease that is natively expressed as part of a polyprotein with a second protein, the second protein having a biological activity which is catalyzed by cleavage of the polyprotein by the protease, said kit comprising:
An in vitro protease assay kit for a protease of the type that is natively expressed as part of a polyprotein with a second protein, the second protein having a biological activity which is catalyzed by cleavage of the polyprotein by the protease, said assay kit comprising:
An in vitro method for evaluating the efficacy of a drug against a biologically active protease chosen from a wild-type protease and a mutant form of a protease, the protease being natively expressed as part of a polyprotein that includes a second protein whose biological activity is dependent upon cleavage of the polyprotein by the protease, said method comprising the steps of:
Dependencies are inferred from the claim text.