Jean-Paul Leonetti

Antiviral activity of conjugates between poly(l-lysine) and synthetic oligodeoxyribonucleotides ☆

Short (14 to 20-mer range) synthetic oligodeoxyribonucleotides (oligos) allow to modulate specifically viral or cellular gene expression at various stages thus providing a versatile tool for fundamental studies and a rational approach to antiviral chemotherapy. Several problems, such as metabolic stability and efficient cell internalization of oligos, still limit this approach appreciably, as briefly discussed here. We demonstrate here that the conjugation of 15-mer (beta)-anomeric oligos to poly(L-lysine) allows a specific protection of various cell lines against vesicular stomatitis virus infection at concentrations lower than 1 microM. This can be achieved with oligos complementary to the viral N-protein mRNA initiation site or to viral intergenic sequences, i.e., to untranscribed regions. No antiviral activity can be obtained with (alpha)-anomeric oligos directed against the same targets, although such analogues are much more resistant to nuclease degradation and form stable hybrids, at least in cell-free experiments.

Resistance to rifampicin: at the crossroads between ecological, genomic and medical concerns.

The first antibiotic of the ansamycin family, rifampicin (RIF), was isolated in 1959 and was introduced into therapy in 1962; it is still a first-line agent in the treatment of diseases such as tuberculosis, leprosy and various biofilm-related infections. The antimicrobial activity of RIF is due to its inhibition of bacterial RNA polymerase (RNAP). Most frequently, bacteria become resistant to RIF through mutation of the target; however, this mechanism is not unique. Other mechanisms of resistance have been reported, such as duplication of the target, action of RNAP-binding proteins, modification of RIF and modification of cell permeability. We suggest that several of these alternative resistance strategies could reflect the ecological function of RIF, such as autoregulation and/or signalling to surrounding microorganisms. Very often, resistance mechanisms found in the clinic have an environmental origin. One may ask whether the introduction of the RIF analogues rifaximin, rifalazil, rifapentine and rifabutin in the therapeutic arsenal, together with the diversification of the pathologies treated by these molecules, will diversify the resistance mechanisms of human pathogens against ansamycins.

The transcription inhibitor lipiarmycin blocks DNA fitting into the RNA polymerase catalytic site

Worldwide spreading of drug‐resistant pathogens makes mechanistic understanding of antibiotic action an urgent task. The macrocyclic antibiotic lipiarmycin (Lpm), which is under development for clinical use, inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Using genetic and biochemical approaches, we show that Lpm targets the σ70 subunit region 3.2 and the RNAP β′ subunit switch‐2 element, which controls the clamping of promoter DNA in the RNAP active‐site cleft. Lpm abolishes isomerization of the ‘closed’‐promoter complex to the transcriptionally competent ‘open’ complex and blocks σ70‐stimulated RNA synthesis on promoter‐less DNA templates. Lpm activity decreases when the template DNA strand is stabilized at the active site through the interaction of RNAP with the nascent RNA chain. Template DNA‐strand fitting into the RNAP active‐site cleft directed by the β′ subunit switch‐2 element and the σ70 subunit region 3.2 is essential for promoter melting and for de novo initiation of RNA synthesis, and our results suggest that Lpm impedes this process.

Antiviral activity of antisense oligonucleotides linked to poly(L-lysine):targets on genomic RNA and/or mRNA of Vesicular Stomatitis Virus

Abstract Synthetic oligonucleotides provide a rational approach to viral genes control. Conjugation of antisense oligodeoxyribonucleotides (directed against several viral sequences) to poly(L-lysine) brings about specific protection against Vesicular Stomatitis Virus at concentrations lower than 1 uM.

Interferons and oncogenes in the control of cell growth and differentiation: working hypothesis and experimental facts.

This short review summarizes available evidence for (i) growth regulatory properties of exogenous as well as recently described autocrine IFNs, (ii) down-regulation of cellular oncogene expression with emphasis on c-myc and (iii) the possible involvement of the IFN-regulated 2-5A pathway at these levels. Initially described as a part of the IFN-induced antiviral mechanism, this double-stranded RNA-activated pathway leads to the preferential degradation of viral mRNAs in IFN-treated virus-infected cells probably through localized activation at the site of virus replication. Such mechanisms could be involved in the regulation of the stability of rapidly turning over mRNAs as for instance c-myc mRNA in IFN-treated cells. Whatever the elegance of the concept, however, experimental evidence is essentially circumstantial; tools developed in our group to strengthen the demonstration are briefly described.

Poly (L Lysine) Mediated Delivery of Nucleic Acids

The concept of “complementary addressed oligonucleotides” as a tool to control specifically gene expression has been initially formulated by the group of Knorre and Vlassov as review in (1985). A first demonstration of a possible control of Rous sarcoma virus gene expression by synthetic oligonucleotides has been provided by Zamecnik and Stephenson (1978).

Poly(L-Lysine) Conjugation: An Efficient Tool for the Introduction of Biologically Active Oligonucleotides in Intact Cells

Gene expression regulation is essentially exerted through specific interactions between nucleic acids sequences or through specific recognition between nucleic acids and proteins. Efficient tools for the introduction in intact cells and tissues of adequately chosen riboor deoxyribonucleotidic sequences have thus been searched. They indeed offer the potential of interfering with the expression of normal genes to study their role in cell metabolism or of attenuating the expression of deleterious genes, as for instance viral genes or deregulated oncogenes. With the exception of plasmids and viruses,’ little success has been achieved yet in the conception of efficient oligonucleotides delivery systems. We demonstrate here that the chemical conjugation of oligonucleotides to poly(L4ysine) (PLL) allows their efficient internalization in intact cells. As outlined in FIGURE 1, periodate-oxidized oligoribonucleotides can be conjugated to PLL-amino groups through reductive amination.* The introduction of a Nrnorpholine ring at oligonucleotides 3’ end also stabilizes them against phosphodiesterases.* This procedure allows the conjugation of oligonucleotides to exposed amino groups in any natural or synthetic polypeptides, thus allowing (at least in principle) the construction of adducts providing both transmembrane transfer and cell targeting. As a first example of the validity of this concept, oligoribonucleotides in the (2’-5’) (A), series were conjugated to PLL. As expected from known intracellular mediators of interferon antiviral action, these (2’-5’) (A), conjugates significantly protect cultured cells from vesicular stomatitis virus (VSV) infection. (TABLE 1 ) . This antiviral activity is due to the activation of RNase L, the specific target of (2’-5’) (A)”, by delivered (2’-5’) (A), oligonucleotides. Indeed, (7-5’) (A),-PLL conjugates produce transient inhibition of protein synthesis and rRNAs cleavages, characteristic of RNase L activation.* PLL conjugation also allowed us to internalize efficiently “antisense” oligodeoxyribonucleotides.’ In these studies, the oligonucleotide sequence was designed to hybridize specifically to the ribosome-binding site of the rnRNA coding for the N protein of VSV. This conjugate exerted a reasonably stable and powerful antiviral activity when added to the culture medium prior VSV infection (TABLE 1). The inhibition appeared highly specific since neither the synthesis of cellular proteins nor the multiplication of encephalomyocarditis virus were affected ~ignificantly.~ Synthetic polypeptides as PLL thus appear as an efficient tool to deliver conjugated oligonucleotides to intact cells a t least in vitro. Preliminary experiments also indicated that (2’-5’) (A), asialofetuin conjugates (FIG. 1 ) provide an antiviral activity in hepatoma cells (HepG2) carrying galactose receptors a t their surface.