The Latency-Associated Nuclear Antigen (LANA) is required for latent replication and persistence of Kaposi´s Sarcoma-associated Herpesvirus (KSHV)/human herpesvirus-8 (HHV-8). It acts via replicating and tethering the virus episome to the host chromatin and exert other functions. We conceived a new approach for the discovery of antiviral drugs to inhibit the interaction between LANA and the viral genome. We applied a biophysical screening cascade and identified the first LANA binders from small, structurally diverse compound libraries. Starting from a fragment-sized scaffold, we generated optimized hits via fragment growing using a dedicated fluorescence polarization-based assay as the structure-activity-relationship driver. We improved compound potency to the double-digit micromolar range. Importantly, we qualified the resulting hit through orthogonal methods employing EMSA, STD-NMR and MST methodologies. This optimized hit provides an ideal starting point for subsequent hit-to-lead campaigns providing evident target-binding, suitable ligand efficiencies and favorable physicochemical properties.
With the aim to develop novel antiviral agents against Kaposi's Sarcoma Herpesvirus (KSHV) we are targeting the latency-associated nuclear antigen (LANA). This protein plays an important role for the viral genome maintenance during latent infection. LANA has the ability to tether the viral genome to the host nucleosomes and, thus, ensures latent persistence of the viral genome in the host cells. By inhibition of the LANA-DNA interaction, we seek to eliminate or reduce the load of the viral DNA in the host. To achieve this goal, we screened our in-house library using a dedicated fluorescence polarization (FP)-based competition assay, which allows for the quantification of the LANA-DNA-interaction inhibition by small organic molecules. We successfully identified three different compound classes capable of disrupting this protein-nucleic acid interaction. We characterized these compounds by IC 50 dose-response evaluation and confirmed the compound-LANA interaction using surface plasmon resonance (SPR) spectroscopy. Furthermore, two of the three hit scaffolds showed only marginal cytotoxicity in two human cell lines. Finally, we conducted STD-NMR competition experiments with our new hit compounds and a previously described fragment-sized inhibitor. Based on these results, future compound linking approaches could serve as a promising strategy for further optimization studies in order to generate highly potent KSHV inhibitors.
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