Inhibition of herpes simplex virus helicase UL9 by netropsin derivatives and antiviral activities of bis-netropsins

Data obtained show that antiviral activities of bis-linked netropsin derivatives are targeted by specific complexes formed by helicase UL9 of herpes simplex virus type 1 with viral DNA replication origins, represented by two OriS sites and one OriL site. According to the results of footprinting studies, bis-netropsins get bound selectively to an A + T cluster which separates interaction sites I and II for helicase UL9 in OriS. Upon binding to DNA, bis-netropsins stabilize a structure of the A + T cluster and inhibit thermal fluctuation-induced opening of AT base pairs which is needed for local unwinding of DNA by helicase UL9. Kinetics of ATP-dependent DNA unwinding in the presence and absence of Pt-bis-netropsin are studied by measuring the efficiency of Forster resonance energy transfer (FRET) between the fluorescent probes attached covalently to 3′- and 5′-ends of the oligonucleotides in the minimal OriS duplex. Pt-bis-netropsin and related molecules inhibit unwinding of OriS duplex by helicase UL9. Pt-bis-netropsin is also able to reduce the rate of unwinding of the AT-rich hairpin formed by the upper strand in the minimal OriS duplex. The antiviral activities and toxicity of bis-linked netropsin derivatives are studied in cell cultured experiments and experiments with animals infected by herpes virus.