10th Conference on Retroviruses and Opportunistic Infections Boston, MA USA - February 10 -14, 2003

Conf Retroviruses Opportunistic Infect 2003 Feb 10-14;10th: abstract no. 9
Z. Debyser¹, C. Pannecouque¹, W. Pluymers¹, B. Van Maele¹, J. Vercammen², V. Tetz³, Y. Engelborghs², E. De Clercq¹, M. Witvrouw^1
1Rega Inst for Med Res, Katholieke Univ Leuven, Belgium; ²Lab for Biomolecular Dynamics, Katholieke Universiteit Leuven, Belgium; and ³St Petersburg Pavlov State Med Univ, Saint-Petersburg, Russia

BACKGROUND: To improve the existing combination therapies and to cope with HIV strains that are resistant to multiple drugs, we searched for effective inhibitors of HIV integrase, the enzyme responsible for inserting the viral cDNA into the host cell chromosome.

METHODS: Selective inhibitors of HIV replication in cell culture were identified by the MT-4/MTT assay. The antiviral activity was determined against a wide range of virus strains. The following mechanism of action studies were performed: oligonucleotide-based integrase (IN) assays, reverse transcriptase assays, time-of-addition experiments, HIV-1 vector transductions followed by real-time PCR quantification of integration, and fluorescence fluctuation analysis of IN-DNA interactions.

RESULTS: A series of 5H-pyrano[2,3-d:-6,5-d']dipyrimidines (PDPs) was identified that blocked the replication of various strains of HIV-1 and HIV-2. The most potent congener 5-(4-nitrophenyl)-2,8-dithiol-4,6-dihydroxy-5H-pyrano [2,3-d:-6,5-d'] dipyrimidine (V-165) inhibited the replication of HIV-1(III_B) in MT‑4 cells at a 50% effective concentration (EC₅₀) of 8.9 µM, that is 14-fold below its cytotoxic concentration. V-165 was equally active against virus strains that were resistant towards inhibitors of viral entry or reverse transcriptase. An HIV-1 strain, resistant to the diketo compounds L-708,906 and S‑1360 retained sensitivity to inhibition by V-165. In combination regimens in cell culture, V-165 acted subsynergistically with zidovudine and nelfinavir and synergistically with nevirapine. V-165 inhibited both reverse transcriptase and integrase activities in enzymatic assays at micromolar concentrations, but a close correlation was found only between the antiretroviral activity observed in cell culture and the inhibitory activity in integrase strand transfer assays. Time-of-addition experiments indicated that V-165 interfered with the viral replication cycle at a time point coinciding with the time of retroviral DNA integration. Quantitative Alu-PCR corroborated that the anti-HIV activity of V-165 is based upon the inhibition of proviral DNA integration. Results obtained by fluorescence fluctuation analysis revealed that inhibition of HIV integrase is due to inhibition of IN-DNA complex formation.

CONCLUSIONS: Based on their mode of action, e.g., inhibition of an HIV integration step that is different from both clinically approved anti-HIV drugs as well as other IN inhibitors such as the diketo acids, PDPs can be considered as candidate drugs to be further pursued in their own right and in drug combination regimens for the therapy of HIV infections.

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