A simple, continuous fluorometric assay for HIV protease

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Abstract
Novel fluorogenic substrates for human immunodeficiency viral protease have been developed based on the principle of fluorescence energy transfer. Starting from a p24/p15 cleavage site-derived hexapeptide substrate, Ac-Thr-Ile-Nle-Nle-Gln-Arg-NH2, incorporation of 2-aminobenzoic acid in place of the acetyl group as the donor and p-NO2-Phe at the P1′ position as acceptor gave the intramolecularly quenched fluorogenic substrate. Cleavage of the substrate by HIV protease released the fluorescent N-terminal tripeptide from its close apposition to the quenching nitrobenzyl group, resulting in enhanced fluorescence. An automated assay based on 96=well microtiter plates and a fluorometric plate reader have been developed, which allow high throughput of compounds in the search for HIV protease inhibitors.