The human immunodeficiency virus type one integrase (HIV-1 integrase) is required for integration of a double-stranded DNA copy of the viral RNA genome into a host chromosome and for HIV replication. We have previously reported that phenolic moieties in compounds such as flavones, caffeic acid phenethyl ester (CAPE), tyrphostins, and curcumin confer inhibitory activity against HIV-1 integrase. We have investigated the actions of several recently described protease inhibitors, possessing novel structural features, on HIV-1 integrase. NSC 158393, which contains four 4-hydroxycoumarin residues, was found to exhibit antiviral, antiprotease, and antiintegrase activity. Both the DNA binding and catalytic activities (3'-processing and strand transfer) of integrase were inhibited at micromolar concentrations. Disintegration catalyzed by an integrase mutant containing only the central catalytic domain was also inhibited, indicating that the binding site for these compounds resides in the central 50-212 amino acids of HIV-1 integrase. Binding at or near the integrase catalytic site was also suggested by a global inhibition of the choice of attacking nucleophile in the 3'-processing reaction. NSC 158393 inhibited HIV-2, feline, and simian immunodeficiency virus integrases while eukaryotic topoisomerase I was inhibited at higher concentrations, suggesting selective inhibition of retroviral integrases. Molecular modeling studies revealed that the two hydroxyls and two carbonyl moieties in NSC 158393 may represent essential elements of the pharmacophore. Antiviral efficacy was observed with NSC 158393 derivatives that inhibited both HIV protease and integrase, and the most potent integrase inhibitors also inhibited HIV protease. Hydroxycoumarins may provide lead compounds for development of novel antiviral agents based upon the concurrent inhibition of two viral targets, HIV-1 integrase and protease.