RESUMEN
The Latency-associated nuclear antigen (LANA) plays a central role for the latent persistence of the Kaposi's Sarcoma Herpesvirus (KSHV) in the human host and helps to establish lifelong infections. Herein, we report our efforts towards hit-to-lead generation starting from a previously discovered LANA-DNA inhibitor. By tethering the viral genome to the host nucleosomes, LANA ensures the segregation and persistence of the viral DNA during mitosis. LANA is also required for the replication of the latent viral episome during the S phase of the cell cycle. We aim to inhibit the interaction between LANA and the viral genome to prevent the latent persistence of KSHV in the host organism. Medicinal chemistry-driven optimization studies and structure-activity-relationship investigation led to the discovery of an improved LANA inhibitor. The functional activity of our compounds was evaluated using a fluorescence polarization (FP)-based interaction inhibition assay and electrophoretic mobility shift assay (EMSA). Even though a crystal structure of the ligand protein complex was not available, we successfully conducted hit optimization toward a low micromolar protein-nucleic acid-interaction inhibitor. Additionally, we applied STD-NMR studies to corroborate target binding and to gain insights into the binding orientation of our most potent inhibitor, providing opportunities for further rational design of more efficient LANA-targeting anti KSHV agents in future studies.
Asunto(s)
Antivirales/farmacología , Infecciones por Herpesviridae/tratamiento farmacológico , Herpesvirus Humano 8/efectos de los fármacos , Isoquinolinas/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Triazoles/farmacología , Antígenos Virales/metabolismo , Antivirales/síntesis química , Antivirales/química , ADN Viral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Infecciones por Herpesviridae/metabolismo , Isoquinolinas/síntesis química , Isoquinolinas/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Proteínas Nucleares/metabolismo , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/químicaRESUMEN
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 in 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 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 IC50 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.
Asunto(s)
Antivirales/farmacología , Herpesvirus Humano 8/efectos de los fármacos , Proteínas Nucleares/antagonistas & inhibidores , Antígenos Virales/metabolismo , Antivirales/toxicidad , ADN/metabolismo , Descubrimiento de Drogas , Células HEK293 , Células Hep G2 , Humanos , Pruebas de Sensibilidad Microbiana , Proteínas Nucleares/metabolismo , Unión Proteica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/toxicidadRESUMEN
The latency-associated nuclear antigen (LANA) is required for latent replication and persistence of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8. It acts via replicating and tethering the virus episome to the host chromatin and exerts 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.