Biochemical characterization of the HIV-1 integrase 3'-processing activity and its inhibition by phosphorothioate oligonucleotides.

To better understand HIV-1 integrase (IN) functions, we determined the kinetic parameters of the 3'-processing reaction. Steady-state kinetic analysis performed using Dixon plots indicated that the concentration of active enzyme was 10-fold lower than that calculated by protein determination. The turnover number was low, suggesting that IN remained bound to DNA after cleavage. The catalytic efficiency increased 10-fold from 30 to 37 degrees C and 2-fold from 37 to 42 degrees C. In enzyme assays carried out at 37 degrees C, both single- and double-stranded phosphorothioate oligos bound to IN with an efficiency comparable to that of the phosphodiester duplex substrate. The competition efficiency of single-stranded oligos was directly related to the sequence length. On the other hand, phosphorothioate duplex U5 LTRs modified in the plus strand were capable of both competing with the substrate and directly inhibiting the 3'-processing activity. These results suggest that, in addition to other modes of action (inhibition of gp120-CD4 interaction and reverse transcriptase), phosphorothioate hetero- and homopolimeric oligos also potently inhibit the IN activity.

Antitumor activity of 4,5,6,7-tetrathiocino (1,2-b: 3,4-b') diimidazolyl-1,3,8,10-tetrasubstituted-2,9-dithiones (R4-todit).

A series of derivatives belonging to a new class of compounds (R4-todit) were highly cytotoxic to a panel of leukaemia- and solid tumour-derived cell lines (IC50 = 0.06-20 microM). The most potent compound was the butyl4 derivative (IC50 = 0.06-5.1 microM); T leukaemia and melanoma cells were the most susceptible cells to this inhibitor (IC50 0.06 microM and 0.1 microM, respectively). The effect of butyl4-todit was irreversible, and led to progressive cell death. The compound showed a comparable potency against exponentially growing and stationary phase cells, and against cell lines expressing the MDR phenotype. The cytotoxicity of butyl4-todit in human normal PBL was up to 20 fold lower than that shown against T leukaemia cells. When tested for antiangiogenic activity in vivo, 1.5 mg/Kg butyl4-todit resulted in over 70% inhibition of the angiogenesis process induced in mice by Kaposi's sarcoma cell secreted products.