Isolation of two highly potent and non-toxic inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase from Salvia miltiorrhiza

Water soluble extracts of the herbal plant, Salvia miltiorrhiza (Danshen) exhibited potent effect against HIV-1 integrase activity in vitro and viral replication in vivo. We have developed an extensive purification scheme to isolate effective, non-toxic inhibitors against human immunodeficiency virus type 1 (HIV-1) using the 3'-processing activity of integrase as a purification guide and assay. Two water soluble compounds, M,22 and M,32, have been discovered by isolating them from S. miltiorrhiza roots in purities of >99.5%, as shown by NMR spectral analysis with yields of 0.018 and 0.038%, respectively. Structural determination revealed that M(5)22 is lithospermic acid and M(5)32 is lithospermic acid B. These two structurally related compounds are potent anti-HIV inhibitors and showed no cytotoxicity to H9 cells at high concentrations (CC100 > 297 muM for M(5)22 and > 223 muM for M(5)32). The IC50 for inhibition of 3'-processing by HIV-1 integrase was found to be 0.83 muM for M(5)22 and 0.48 muM for M(5)32. In addition, M(5)22 and M(5)32 inhibited HIV-1 integrase catalytic activities of 3'-joining to the target DNA with IC50 of 0.48 muM for M(5)22 and 0.37 muM for M(5)32. Furthermore, kinetic and mechanistic studies suggested that drug binding to HIV-1 integrase and inhibition of enzymatic activity occur at a fast rate. Both M(5)22 and M(5)32 do not prevent HIV entry in H9 cells. They also show no inhibition of reverse transcriptase activity in infected cells. The levels of intracellular strong stop and full-length viral DNA remained unchanged following drug treatment. However, both inhibitors strongly suppressed the acute HIV-1 infection of H9 cells with IC50 values of 2 and 6.9 muM for M(5)22 and M(5)32, respectively. Thus these two selective integrase inhibitors hold promise as a novel class of therapeutic drugs for AIDS based on their high potencies and absence of cytotoxicity. (c) 2002 Elsevier Science B.V. All rights reserved.