Because of the specificity of Watson-Crick base pairing, attempts are now being made to use oligodeoxynucleotides (oligos) in the therapy of human disease. However, for a successful outcome, the oligo must meet at least six criteria: (i) the oligos can be synthesized easily and in bulk; (ii) the oligos must be stable in vivo; (iii) the oligos must be able to enter the target cell; (iv) the oligos must be retained by the target cell; (v) the oligos must be able to interact with their cellular targets; and (vi) the oligos should not interact in a non-sequence-specific manner with other macromolecules. Phosphorothioate oligos are examples of oligos that are being considered for clinical therapeutic trials and meet some, but not all, of these criteria. The potential use of phosphorothioate oligos as inhibitors of viral replication is highlighted.

Several Z-and E-methylenecyclopropane nucleoside analogues were synthesized and evaluated for antiviral activity. Reaction of the Z-and E-2-amino-6-chloropurine methylenecyclopropanes with ammonia or cyclopropylamine gave 2,6-diamino or 2-amino-6-cyclopropylamino analogues. Alkylation elimination of N 4 -acetylcytosine with ethyl Z-and E-2-bromo-2-bromomethylcyclopropane-1-carboxylates gave a mixture of the Zand E-methylenecyclopropane derivatives of cytosine. Reduction furnished a mixture of syncytol and the E isomer. Benzoylation led to the respective N 4 -benzoyl derivatives which were separated by chromatography. Debenzoylation afforded pure syncytol and the E isomer. Alkylation of 2,4-bis-O-trimethylsilylthymine with ethyl Z-and E-2-bromo-2-bromomethylcyclopropane-1-carboxylates gave the corresponding Z-and E-1-bromocyclopropylmethylderivatives of thymine. Basecatalysed elimination of HBr gave Z-and E-methylenecyclopropane carboxylic esters. Reduction furnished, after chromatographic separation, synthymol and the E isomer. The Z/E isomeric assignment of the obtained products followed from 1 H NMR spectroscopy. The methylenecyclopropane analogues were tested for antiviral activity in vitro against human and murine cytomegalovirus (HCMV, MCMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV), hepatitis B virus (HBV), herpes simplex virus types 1 and 2 (HSV-1, HSV-2), human herpesvirus 6 (HHV-6) and human immunodeficiency virus type 1 (HIV-1). The Z-2-amino-6-cyclopropylaminopurine analogue was the most effective agent against HCMV (EC 50 or EC 90 0.4-2 µM) followed by syncytol and the Z-2,6-diaminopurine analogues (EC 50 or EC 90 3.4-29 and 11-24 µM, respectively). The latter compound was also a strong inhibitor of MCMV (EC 50 0.6 µM). Syncytol was the most potent against EBV (EC 50 <0.41 and 2.5 µM) followed by the Z-2,6-diaminopurine (EC 50 1.5 and 6.9 µM) and the Z-2-amino-6-cyclopropylaminopurine derivative (EC 50 11.8 µM). Syncytol was also most effective against VZV (EC 50 3.6 µM). Activity against HSV-1, HSV-2 and HHV-6 was generally lower; synthymol had an EC 50 of 2 µM against HSV-1 (ELISA) and 1.3 µM against EBV in Daudi cells but was inactive in other assays. The 2-amino-6-cyclopropylamino analogue displayed EC 50 values between 215 and >74 µM in HSV-1 and HSV-2 assays. 2-Amino-6-cyclopropylaminopurine and 2,6-diaminopurine derivatives were effective against HBV (EC 50 2 and 10 µM, respectively), whereas none of the analogues inhibited HIV-1 at a higher virus load. Syncytol and the E isomer were equipotent against EBV in Daudi cells but the E isomer was much less effective in DNA hybridization assays. The E-2,6-diaminopurine analogue and E isomer of synthymol were devoid of antiviral activity.

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Epstein-Barr virus (EBV) is the cause of infectious mononucleosis and is associated with three human malignancies. Acyclovir [9-(2-hydroxyethoxymethyl)guanine], the first clinically useful drug effective against replication of EBV, is without effect against latent or persistent EBV infection. Three nucleoside analogs, E-5-(2-bromovinyl)-2'-deoxyuridine, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine, and 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil are potent inhibitors of EBV replication in vitro. Moreover, in contrast to the reversibility of viral inhibition by Acyclovir, these three drugs have prolonged effects in suppressing viral replication even after the drugs are removed from persistently infected cell cultures.

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Vascular endothelial growth factor A (VEGF-A) is a pivotal player in angiogenesis. It is capable of influencing such cellular processes as tubulogenesis and vascular smooth muscle cell (VSMC) proliferation, yet very little is known about the actual signaling events that mediate VEGF-A induced VSMC phenotypic switch. In this report, we describe the identification of an intricate VEGF-A-induced signaling cascade that involves VEGFR2, STAT3, and Myocardin. We demonstrate that VEGF-A promotes VSMC proliferation via VEGFR2/STAT3-mediated upregulating the proliferation of markers like Cyclin D1 and PCNA. Specifically, VEGF-A leads to nitrosylation of Myocardin, weakens its effect on promoting the expression of contractile markers and is unable to inhibit the activation of STAT3. These observations reinforce the importance of nitric oxide and S-nitrosylation in angiogenesis and provide a mechanistic pathway for VEGF-A-induced VSMC phenotypic switch. In addition, Myocardin, GSNOR and GSNO can create a negative feedback loop to regulate the VSMC phenotypic switch. Thus, the discovery of this interactive network of signaling pathways provides novel and unexpected therapeutic targets for angiogenesis-dependent diseases.

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