Audrey Di Giorgio

Fluorescent labeling of human mesenchymal stem cells by thiophene fluorophores conjugated to a lipophilic carrier

Fluorescent labeling of human mesenchymal stem cells (hMSCs) is essential for their study in vitro and in vivo. Here, we report a new chemical biodelivery method for labeling hMSCs using a series of very efficient and highly versatile thiophene-based fluorescent dyes. Such conjugates contribute to a stable and viable labeling of hMSCs and constitute a promising strategy for in vivo applications.

Short peptide nucleic acids (PNA) inhibit hepatitis C virus internal ribosome entry site (IRES) dependent translation in vitro.

The internal ribosome entry site (IRES) of hepatitis C virus (HCV) which governs the initiation of protein synthesis from viral RNA represents an ideal target for antisense approaches. Using an original bicistronic plasmid, we first established that sequence and translational activity of HCV IRESs cloned from six patients, whether responders or not to combination therapy, were conserved. We then tested the hypothesis that antisense molecules, i.e. short peptide nucleic acids (PNA), could inhibit HCV translation by binding to the highly conserved IIId or IV loop regions of the IRES. Five 6-10mer PNAs were designed. They strongly inhibit HCV IRES-driven translation in a rabbit reticulocyte lysate assay. This inhibition was highly specific since corresponding PNAs with only one mismatch were inactive. Short phosphorothioate oligonucleotides of same sequence were unable to inhibit HCV translation. PNA molecule was shown to have anti-HCV activity in Huh-7.5 cells when electroporated with a full-length HCV genome construct. Using oligonucleotide as carrier, PNA was also transfected in HCV replicon-harboring cells and in JFH1 infected Huh-7.5 cells.

Thermodynamic studies of a series of homologous HIV-1 TAR RNA ligands reveal that loose binders are stronger Tat competitors than tight ones

RNA is a major drug target, but the design of small molecules that modulate RNA function remains a great challenge. In this context, a series of structurally homologous ‘polyamide amino acids’ (PAA) was studied as HIV-1 trans-activating response (TAR) RNA ligands. An extensive thermodynamic study revealed the occurence of an enthalpy–entropy compensation phenomenon resulting in very close TAR affinities for all PAA. However, their binding modes and their ability to compete with the Tat fragment strongly differ according to their structure. Surprisingly, PAA that form loose complexes with TAR were shown to be stronger Tat competitors than those forming tight ones, and thermal denaturation studies demonstrated that loose complexes are more stable than tight ones. This could be correlated to the fact that loose and tight ligands induce distinct RNA conformational changes as revealed by circular dichroism experiments, although nuclear magnetic resonance (NMR) experiments showed that the TAR binding site is the same in all cases. Finally, some loose PAA also display promising inhibitory activities on HIV-infected cells. Altogether, these results lead to a better understanding of RNA interaction modes that could be very useful for devising new ligands of relevant RNA targets.

An Efficient Biodelivery System for Antisense Polyamide Nucleic Acid (PNA)

With the aim of developing a general and straightforward procedure for the intracellular delivery of naked peptide nucleic acids (PNAs), we designed an intracellularly biodegradable triphenylphosphonium (TPP) cation based transporter system. In this system, TPP is linked, via a biolabile disulfide bridge, to an activated mercaptoethoxycarbonyl moiety, allowing its direct coupling to the N-terminal extremity of a free PNA through a carbamate bond. We found that such TPP-PNA-carbamate conjugates were highly stable in a cell culture medium containing fetal calf serum. In a glutathione-containing medium mimicking the cytosol, the conjugates were rapidly degraded into an unstable intermediate, which spontaneously decomposed, releasing the free PNA. Using a fluorescence-labeled PNA-TPP conjugate, we demonstrated that conjugates were taken up by cells. Efficient cellular uptake and release of the PNA into the cytosol was further confirmed by the anti-HIV activity measured for the TPP-conjugate of a 16-mer PNA targeting the TAR region of the HIV-1 genome. This conjugate exhibited an IC(50) value of 1 microM, while the free 16-mer PNA did not inhibit replication of HIV in the same cellular test.

Oncogenic MicroRNAs Biogenesis as a Drug Target: Structure–Activity Relationship Studies on New Aminoglycoside Conjugates

MicroRNAs (miRNAs) are a recently discovered category of small RNA molecules that regulate gene expression at the post-transcriptional level. Accumulating evidence indicates that miRNAs are aberrantly expressed in a variety of human cancers and that the inhibition of these oncogenic miRNAs could find application in the therapy of different types of cancer. Herein, we describe the synthesis and biological evaluation of new small-molecule drugs that target oncogenic miRNAs production. In particular, we chose to target two miRNAs (i.e., miRNA-372 and -373) implicated in various types of cancer, such as gastric cancer. Their precursors (pre-miRNAs) are overexpressed in cancer cells and lead to mature miRNAs after cleavage of their stem-loop structure by the enzyme Dicer in the cytoplasm. Some of the newly synthesized conjugates can inhibit Dicer processing of the targeted pre-miRNAs in vitro with increased efficacy relative to our previous results (D.D. Vo et al., ACS Chem. Biol. 2014, 9, 711-721) and, more importantly, to inhibit proliferations of adenocarcinoma gastric cancer (AGS) cells overexpressing these miRNAs, thus representing promising leads for future drug development.

Polyamide Amino Acids trimers as TAR RNA ligands and anti-HIV agents

Based on a split-and-mix strategy, a library of trimeric Polyamide Amino Acids (PAA) incorporating four different amino acids (Lys, Ala, Arg, and Phe) has been prepared. Screening of the batches for HIV TAR RNA binding in a fluorescent assay allowed the identification of several components that interact with TAR RNA at a micromolar concentration, with a good TAR versus tRNA specificity. Some of these compounds compete efficiently with the association of TAR and Tat protein. In cell cultures, these compounds display a moderate antiviral activity, associated nevertheless with some toxicity. Overall, these results confirm that this new family can be a basis for the design of novel RNA targeting drugs.

Small-molecule approaches toward the targeting of oncogenic miRNAs: roadmap for the discovery of RNA modulators

miRNAs are a recently discovered class of small noncoding RNAs implicated in the regulation of gene expression. The deregulation of miRNAs levels has been linked to the development of various cancers where oncogenic miRNAs are overexpressed and tumor suppressor miRNAs are underexpressed. Here we report the three main strategies developed in order to discover small-molecule drugs able to selectively interfere with oncogenic miRNAs: the high throughput screening of large libraries of compounds, the focused screening of small libraries of molecules that are known to be able to interact with RNA thus being supposed modulators of miRNAs pathway and the design of small molecules based on the secondary structure of targeted RNA and/or three-dimensional structure of enzymes involved in miRNAs pathway.

Inhibition of HIV Replication by Cyclic and Hairpin PNAs Targeting the HIV-1 TAR RNA Loop.

Human immunodeficiency virus-1 (HIV-1) replication and gene expression entails specific interaction of the viral protein Tat with its transactivation responsive element (TAR), to form a highly stable stem-bulge-loop structure. Previously, we described triphenylphosphonium (TPP) cation-based vectors that efficiently deliver nucleotide analogs (PNAs) into the cytoplasm of cells. In particular, we showed that the TPP conjugate of a linear 16-mer PNA targeting the apical stem-loop region of TAR impedes Tat-mediated transactivation of the HIV-1 LTR in vitro and also in cell culture systems. In this communication, we conjugated TPP to cyclic and hairpin PNAs targeting the loop region of HIV-1 TAR and evaluated their antiviral efficacy in a cell culture system. We found that TPP-cyclic PNAs containing only 8 residues, showed higher antiviral potency compared to hairpin PNAs of 12 or 16 residues. We further noted that the TPP-conjugates of the 8-mer cyclic PNA as well as the 16-mer linear PNA displayed similar antiviral efficacy. However, cyclic PNAs were shown to be highly specific to their target sequences. This communication emphasizes on the importance of small constrained cyclic PNAs over both linear and hairpin structures for targeting biologically relevant RNA hairpins.

Deciphering structure-activity relationships in a series of Tat/TAR inhibitors.

A series of pentameric “Polyamide Amino Acids” (PAAs) compounds derived from the same trimeric precursor have been synthesized and investigated as HIV TAR RNA ligands, in the absence and in the presence of a Tat fragment. All PAAs bind TAR with similar sub-micromolar affinities but their ability to compete efficiently with the Tat fragment strongly differs, IC50 ranging from 35 nM to >2 μM. While NMR and CD studies reveal that all PAA interact with TAR at the same site and induce globally the same RNA conformational change upon binding, a comparative thermodynamic study of PAA/TAR equilibria highlights distinct TAR binding modes for Tat competitor and non-competitor PAAs. This led us to suggest two distinct interaction modes that have been further validated by molecular modeling studies. While the binding of Tat competitor PAAs induces a contraction at the TAR bulge region, the binding of non-competitor ones widens it. This could account for the distinct PAA ability to compete with Tat fragment. Our work illustrates how comparative thermodynamic studies of a series of RNA ligands of same chemical family are of value for understanding their binding modes and for rationalizing structure-activity relationships.

AZT and AZT-monophosphate Prodrugs Incorporating HIV-protease Substrate Fragment: Synthesis and Evaluation as Specific Drug Delivery Systems

With the view to deliver anti-HIV nucleoside and nucleoside-monophosphate (MP) analogues specifically into HIV-infected cells, we synthesized a series of ester and phosphoramidate peptide conjugates of zidovudine (AZT) and of AZT-MP, respectively, wherein the peptide sequences derive from a HIV-protease (PR) hydrolysable substrate. Their in vitro stability with respect to hydrolysis, anti-HIV activity and cytotoxicity, and ability to inhibit the HIV-PR activity were investigated. Concerning the ester AZT-peptide conjugates, their antiviral activity level in thymidine kinase-expressing (TK+) CEM-SS and MT-4 cells was in most cases closely correlated to their hydrolysis rate: the faster the hydrolysis, the closer the anti-HIV activity to that of AZT. None of them was a HIV-PR substrate, indicating that their antiviral activity was not related to their intracellular hydrolysis by this enzyme. None of them inhibited HIV in TK-deficient (TK−) CEM cells, demonstrating that they probably act as prodrugs of AZT. Most of the phosphoramidate peptide conjugates of AZT-MP were rapidly degraded in a physiological buffer into several metabolites including AZT. Their anti-HIV activity in TK+ CEM-SS and MT-4 cells was much lower than that of AZT, indicating that only low amounts of AZT or AZT-MP were released into cells during incubation. Antiviral activities measured on TK− CEM cells for some phosphoramidates suggest that low amounts of AZT-MP could be released intracellularly. However, this AZT-MP release was not initiated by a HIV-PR hydrolysis, as no evidence for peptide cleavage was obtained by HPLC analysis of one representative compound after incubation with HIV-PR.