Book of Abstracts: Albany 2007
June 19-23 2007
DNA Binding Properties and Antiviral Activity of two Bis-Netropsins Containing Cis-Diammineplatinum(II) Group in the Linker Between Two Netropsin Moeties
We compared DNA-binding properties and antiviral activities of two bis-netropsins containing cis-diammine Pt(II) groups attached to each netropsin-like fragment via one (Pt-bis-Nt) or two (Pt*-bis-Nt) glycine residues. Our observations show that Pt-bis-Nt and Pt*-bis-Nt bind strongly and selectively to AT-clusters on DNA . However, Pt*-bis-Nt exhibits practically no antiviral activity in Vero cell culture experiments, whereas Pt-bis?Nt strongly inhibits reproduction of herpes simplex virus 1 with the selectivity index equal approximately to 60 . The possible target sites for binding of Pt-bis-Nt and Pt*-bis-Nt are long AT-tracks in the origin of replication (OriS and OriL) of the viral DNA. Flanking the AT-cluster in the OriS are the recognition sites for the origin binding protein (OBP) encoded by the UL9 gene of the herpes virus. Specific and cooperative binding of OBP dimers to these sites leads to a destabilization of AT-cluster . In the presence of ATP and another viral protein ICP8 (single-stranded DNA binding protein) OBP induces unwinding of the minimal OriS duplex (80 bp). Interaction of the bis-netropsin with the AT-track may prevent destabilization of its structure and may interfere with the assembly and normal functioning of the viral proteins UL9 and ICP8. Interaction of Pt-bis-Nt and Pt*-bis-Nt with the fragment of OriS containing AT - cluster was studied by DNase I footprinting and CD spectroscopy. The CD data show that Pt*-bis-Nt interacts with the AT-track predominantly in the extended conformation and self-associated dimer form. In contrast Pt-bis-Nt binds to this fragment both in the extended conformation and parallel-stranded hairpin form. The footprints produced by Pt-bis-Nt and Pt*-bis-Nt at the same concentration level are different. Pt-bis-Nt protects from DNase I cleavage a longer DNA region as compared with the footprint size generated in the presence of Pt*-bis-Nt, and protection effect is observed at lower concentration. In addition Pt-bis-Nt protects from cleavage DNA site with the sequence 5'-TATAGGTATA-3', whereas in the presence of Pt*-bis-Nt no protection in this DNA region is detected. From our previous studies it is known that Pt-bis-Nt in the parallel-stranded hairpin form binds strongly to the DNA site with the sequence 5'-TATAT-3'  which is present in the OriS AT-track. These observations suggest that antiviral activities of these bis ? linked netropsin derivatives can be correlated with their affinity and mode of binding to the AT-track in OriS.
Support for this work from the Program of Russian Academy of Sciences on Molecular and Cell Biology is gratefully acknowledged.
References and Footnotes
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Moscow 119991, Vavilov str. 32, Russia