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DESIGN AND CHARACTERIZATION OF NOVEL INTEGRATION INHIBITORS Virginie Suchaud (1), Fabrice Bailly (1), Cédric Lion (1), Belete A. Desimmie (2), Frauke Christ (2), Zeger Debyser (2), Philippe Cotelle (1) (1) universite de Lille 1, EA 4478, Villeneuve d'Ascq, France; (2) Molecular Medicine, K. U. Leuven and IRC KULAK, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium Background. Tackling the global AIDS epidemic is a goal that to date remains unfulfilled. It is estimated that nearly 40 million people live with HIV, and some 2.5 million become infected each year. While no cure has been found, HIV combination therapy prevents the replication of the virus, thus enabling the patient to remain healthy for many years. There are 4 main classes of HIV antiretroviral drugs, namely fusion/entry inhibitors, protease inhibitors, reverse transcriptase inhibitors, and integrase inhibitors. Indeed, the FDA approval in 2007 of Raltegravir (Isentress®, first strand transfer selective integrase inhibitor) validated the latter enzyme as a major therapeutic target. However, due to the appearance of mutant viruses resistant to Raltegravir, it is vital for the AIDS research community to discover a second generation of integrase inhibitors which would not display Raltegravir cross-resistance. We herein present a family of second generation integrase inhibitors. Methods. From a library of about one eighty compounds, we first identified one particular hit (MB76) displaying original and exciting biological activity: The synthesized molecules have been evaluated as integrase inhibitors (both on the overall activity and on the specific strand transfer activity) and on the viral replication (on MT-4 cells). MB76 has been tested on different HIV-1 and HIV-2 strains on two different cell lines. Eventual cross-resistance has been checked on several mutant viral strains (including raltegravir-resistant mutants). Time of addition experiments have been done to control that the integration is the effective target of MB76. Using the available crystallographic structures of Prototype Foamy Virus Integrase with DNA, divalent ions (Mg or Mn) and inhibitor (raltegravir or elvitegravir), we have developed a molecular docking method which allows us to model the binding mode of our inhibitors, using GOLD™. A second series of inhibitors has then been synthesized. Results: specific findings. MB76 is a strong inhibitor of the overall integrase activity (IC50 = 56 nM). However there is evidence that its mechanism of action is quite different to that of the leading integrase inhibitors (namely Raltegravir, IC50 = 10 nM). Indeed, Raltegravir is a selective strand transfer inhibitor (ST, 7 nM, 3’-P, 900 nM). MB76, on the contrary, shows inhibition of both integrase-catalysed steps (ST 99 nM and 3’-P 66 nM). MB76 inhibits HIV-1 and HIV-2 strains with similar potency and inhibits HIV replication in primary human cells without co-receptor distinction. MB76 exclusively inhibits the integrase catalytic activity during HIV replication and does not present cross-resistance with raltegravir. Time of addition experiments after viral cell infection shows that MB76 does inhibit the integration step. The docking of MB76 in the PFV IN active site allowed us to synthesize a new series of molecules and find a new compound (VS39) superior to MB76. VS39 inhibits the overall (IC50 = 10.6 nM) and the strand transfer (IC50 = 14.5 nM) IN activities. VS39 inhibits the viral replication (EC50 = 103 nM) with a low cytotoxicity (CC50 = 121 µM) leading to a high therapeutic index (>1000). Conclusions. More than one hundred new compounds have been synthesized leading to the discovery of IN inhibitors with an original mode of action. The best drugs of this series, to date, have nanomolar activities on HIV-1 integrase and submicromolar activities on the viral replication. Improvement of the antiviral activities may lead in a next future to a good candidate for preclinical evaluation.