Isolation of two highly potent and non-toxic inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase and viral replication

Water soluble extracts of the herbal plant, Salvia miltiorrhiza (Danshen) exhibited potent effects against HIV-1 integrase activity in vitro and viral replication in vivo. A purification scheme has been developed to isolate effective, non-toxic inhibitors against human immunodeficiency virus type 1 (HIV-1) using the 3′ -end processing activity of integrase as a purification guide and assay. Two water soluble compounds, M₅22 and M₅32, have been discovered by isolating them from Salvia miltiorrhiza roots in purities of >99.5% by NMR spectral analysis and yields of 0.018% and 0.038% respectively. Structural determination revealed that M₅22 is lithospermic acid and M₅32 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 μM for M₅22 and >223 μM for M₅32). The IC₅₀ for inhibition of 3′′-processing by HIV-1 integrase was found to be 0.83 μM for M₅22 and 0.48 μM for M₅ 32. In addition, M₅22 and M₅32 inhibited HIV-1 integrase catalytic activities of 3′-joining to the target DNA with an IC₅₀ of 0.48 μM for M₅22 and 0.37 μM for M₅32. Kinetic and mechanistic studies suggested that drug binding to HIV-1 integrase and inhibition of enzymatic activity occurred at a fast rate. Both M₅22 and M₅32 did not prevent HIV entry in H9 cells. They also showed no inhibition of reverse transcriptase activity in infected cells. The levels of intracellular strong-stop and full-length DNA remained unchanged following drug exposure. However, both inhibitors strongly suppressed the acute HIV-1 infection of H9 cells with five different strains of drug-resistant and primary isolates. M₅22 and M₅32 drugs inhibited three drug-resistant strains of HIV-1 with IC₅₀ values of (1.1–6 μg/ml, 2–11.1 μM), (1–6 μg/ml, 1.3–8.3 μM) respectively. For two primary isolate strains of HIV-1, both M₅22 and M₅32 inhibited replication with IC ₅₀ of (9–11 μg/ml, 16.7–20.4 μM) and (6–7 μg/ml, 8.3–9.7 μM). 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.