Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Specific Protein-DNA Complexes as Platforms for Design of New Types of Antiviral Drugs
A new design strategy is developed for synthesis of sequence specific DNA binding ligands. It is based on modular assembly of pyrrole(imidazole) carboxamides and isohelical pseudopeptides of the form (XY)n where Y is a glycine residue. n is the degree of polymerization, X is an unusual aminoacid residue containing five-membered aromatic ring (such as 4-aminomethylthiazole-2-carboxylic acid residue).
The herpes simplex virus type 1 origin-binding protein is a DNA helicase encoded by the UL9 gene. The protein binds in a sequence-specific manner to the viral origin of replication OriS or OriL. In order to to search for efficient inhibitors of the UL9 activity we have obtained a recombinant UL9 protein expressed in E. coli cells. The UL9 gene has been amplified by PCR and inserted into a modified plasmid pET14 (Novagen) between NdeI and KpnI sites. We have found that purified recombinant UL9 protein binds to Boxes I and II in OriS and possesses DNA helicase and ATPase activities. In the presence of ATP and another viral protein ICP8 (single-stranded DNA binding protein) the initiator protein induces unwinding of the minimal OriS duplex (80 bp). The protein also binds strongly to a single-stranded DNA (OriS*) containing a stable Box I-Box III hairpin and disordered tail at the 3'-end, as observed for the first time by Aslani et al. (4).
Until now, nucleosides related to acyclovir were the only compound class available for systematic treatments of herpes disease. In the present work, new minor groove binding ligands have been synthesized which selectively inhibit development of virus-induced cytopathogenic effect in Vero cell culture infected with herpes simplex virus type 1 and vacinia virus. Studies on binding of these compounds to DNA and synthetic poly- and oligonucleotides have been performed by UV and CD spectroscopy, gel mobility shift assays and DNase I footprinting. Footprinting studies reveal that some of them exhibit strong preferences for binding to the AT-cluster in OriS and protect it from cleavage by DNase I.
The observed antiviral activity of the minor groove binding ligands can be attributed to their abilities to inhibit fluctuation opening of AT-base pairs and DNA bending which is induced upon binding of UL9 protein to the Boxes I and II. We have found that in the presence of bis-linked netropsin derivatives the rate of DNA unwinding by the UL9 protein is reduced. Some of the drugs binds strongly to the intermediate conformation (OriS*) represented by a single-stranded tail at the 3'-end and stable Box I-Box III hairpin. We have compared the 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 experiments show that Pt-bis-Nt and Pt*-bis-Nt bind strongly and selectively to AT-rich regions on DNA. However, Pt*-bis-Nt exhibits practically no antiviral activity in cell culture experiments, whereas Pt-bis?Nt inhibits reproduction of herpes simplex virus type 1 with the selectivity index equal to 60. The CD spectroscopy studies and UV melting experiments show that there are substantial differences in the mode of binding of these ligands to OriS* and the thermostability of the corresponding complexes which can be correlated with their antiviral activities.
Figure : Sequence of the minimal OriS duplex (A). Indicated are the positions of two palindromes and the interaction sites for UL9 dimers (boxes I, II and III). Intermediate active and inactive forms of OriS* suggested by Aslani et al. are shown (B and C). An inactive form (C) is stabilized upon binding of the ligand to the AT-rich hairpin in OriS*.
References and Foonotes