Olivier Chaloin

A simple approach to cancer therapy afforded by multivalent pseudopeptides that target cell-surface nucleoproteins.

Recent studies have implicated the involvement of cell surface forms of nucleolin in tumor growth. In this study, we investigated whether a synthetic ligand of cell-surface nucleolin known as N6L could exert antitumor activity. We found that N6L inhibits the anchorage-dependent and independent growth of tumor cell lines and that it also hampers angiogenesis. Additionally, we found that N6L is a proapoptotic molecule that increases Annexin V staining and caspase-3/7 activity in vitro and DNA fragmentation in vivo. Through affinity isolation experiments and mass-spectrometry analysis, we also identified nucleophosmin as a new N6L target. Notably, in mouse xenograft models, N6L administration inhibited human tumor growth. Biodistribution studies carried out in tumor-bearing mice indicated that following administration N6L rapidly localizes to tumor tissue, consistent with its observed antitumor effects. Our findings define N6L as a novel anticancer drug candidate warranting further investigation.

Monoclonal antibody-targeted, temperature-sensitive liposomes: In vivo tumor chemotherapeutics in combination with mild hyperthermia.

The development of actively targeted, responsive delivery vectors holds great promise for cancer therapy. Here, we investigated whether enhanced therapeutic activity of temperature sensitive liposomes (TSL) could be obtained by mild hyperthermia-triggered release of the chemotherapeutic drug doxorubicin (DOX) after hCTMO1 monoclonal antibody (anti-MUC-1) binding and uptake into cancer cells. We showed that traditional TSL (TTSL) liposome systems maintained their physicochemical and thermal properties after conjugation to hCTMO1 full IgG. Receptor-mediated cellular uptake and cytotoxic efficacy of antibody-targeted TTSL (TTSL-Ab) were investigated using 2D and 3D cell culture models. Significant enhancement in cellular uptake and cytotoxic activity after 1h of heating at 42 °C was observed for TTSL-Ab compared to non-targeted liposomes in MUC-1 over-expressing breast cancer cells (MDA-MB-435). Tissue distribution and in vivo therapeutic activity were studied using different heating protocols to explore the effect of mild hyperthermia on the tumor accumulation of targeted TTSL and their therapeutic effect. Application of local, mild hyperthermia (42°C) significantly increased the tumor accumulation of targeted TSL compared to non-targeted liposomes, associated with a moderate improvement in therapeutic activity and survival.

Peptide-based approaches to treat lupus and other autoimmune diseases

After a long period where the potential of therapeutic peptides was let into oblivion and even dismissed, there is a revival of interest in peptides as potential drug candidates. Novel strategies for limiting metabolism and improve their bioavailability, and alternative routes of administration have emerged. This resulted in a large number of peptide-based drugs that are now being marketed in different indications. Regarding autoimmunity, successful data have been reported in numerous mouse models of autoimmune inflammation, yet relatively few clinical trials based on synthetic peptides are currently underway. This review reports on peptides that show much promises in appropriate mouse models of autoimmunity and describes in more detail clinical trials based on peptides for treating autoimmune patients. A particular emphasis is given to the 21-mer peptide P140/Lupuzor that has completed successfully phase I, phase IIa and phase IIb clinical trials for systemic lupus erythematosus.

The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro

The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44–61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties.

Antibody Covalent Immobilization on Carbon Nanotubes and Assessment of Antigen Binding

Controlling the covalent bonding of antibodies onto functionalized carbon nanotubes is a key step in the design and preparation of nanotube-based conjugates for targeting cancer cells. For this purpose, an anti-MUC1 antibody (Ab) is linked to both multi-walled (MWCNTs) and double-walled carbon nanotubes (DWCNTs) using different synthetic strategies. The presence of the Ab attached to the nanotubes is confirmed by gel electrophoresis and thermogravimetric analysis. Most importantly, molecular recognition of the antigen by surface plasmon resonance is able to determine similar Ab binding capacities for both Ab-DWCNTs and Ab-MWCNTs. These results are very relevant for the design of future receptor-targeting strategies using chemically functionalized carbon nanotubes.

Synthesis of chiral urethane N-alkoxycarbonyl tetramic acids from urethane N-carboxyanhydrides (UNCAs)

Abstract The synthesis of chiral N-protected tetramic acid derivatives which are important precursors of β-hydroxy γ-amino acid under mild conditions is described. Reaction of urethane-N-carboxyanhydrides (UNCAs) with Meldrums acid in the presence of a tertiary amine, followed by subsequent cyclisation produced tetramic acid derivatives. This procedure is applicable to Boc-, Fmoc- and Z- N-carboxyanhydrides.

HIV-1 Tat protein directly induces mitochondrial membrane permeabilization and inactivates cytochrome c oxidase.

The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨm) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor.

The relationship between the diameter of chemically-functionalized multi-walled carbon nanotubes and their organ biodistribution profiles in vivo

Carbon nanotubes (CNTs) exhibit unique properties which have led to their applications in the biomedical field as novel delivery systems for diagnosis and therapy purposes. We have previously reported that the degree of functionalization of CNTs is a key factor determining their biological behaviour. The present study broadens the spectrum by investigating the impact of the diameter of CNTs using two series of multi-walled CNTs (MWNTs) with distinct differences in their diameters. Both MWNTs were doubly functionalized by 1,3-dipolar cycloaddition and amidation reactions, allowing the appended functional groups to be further conjugated with radionuclide chelating moieties and antibodies or antibody fragments. All constructs possessed comparable degree of functionalization and were characterized by thermogravimetric analysis, transmission electron microscopy, gel electrophoresis and surface plasmon resonance. The MWNT conjugates were radio-labelled with indium-111, which thereby enabled in vivo single photon emission computed tomography/computed tomography (SPECT/CT) imaging and organ biodistribution study using γ-scintigraphy. The narrow MWNTs (average diameter: 9.2 nm) demonstrated enhanced tissue affinity including non-reticular endothelial tissues compared to the wider MWNTs (average diameter: 39.5 nm). The results indicate that the higher aspect ratio of narrow MWNTs may be beneficial for their future biological applications due to higher tissue accumulation.

Sandwich ELISA assay for the quantitation of palytoxin and its analogs in natural samples

Palytoxins are potent marine biotoxins that have recently become endemic to the Mediterranean Sea, and are becoming more frequently associated with seafood. Due to their high toxicity, suitable methods to quantify palytoxins are needed. Thus, we developed an indirect sandwich ELISA for palytoxin and 42-hydroxy-palytoxin. An intralaboratory study demonstrated sensitivity (limit of detection, LOD = 1.1 ng/mL; limit of quantitation, LOQ = 2.2 ng/mL), accuracy (bias of 2.1%), repeatability (RSDr = 6% and 9% for intra- and interassay variability, respectively) and specificity: other common marine toxins (okadaic acid, domoic acid, saxitoxin, brevetoxin-3, and yessotoxin) do not cross-react in this assay. It performed well in three different matrices: observed LOQs were 11.0, 9.6, and 2.4 ng/mL for mussel extracts, algal net samples and seawater, respectively, with good accuracy and precision. The LOQ in seafood is 11 μg palytoxin/kg mussel meat, lower than that of the most common detection technique, LC-MS/MS.

The binding affinity of double‐stranded RNA motifs to HIV‐1 Tat protein affects transactivation and the neutralizing capacity of anti‐Tat antibodies elicited after intranasal immunization

In this study we examined the hypothesis that the binding affinity of two double‐stranded (ds) RNA motifs to HIV‐1 Tat protein might affect transactivation and the type of anti‐Tat immune responses. Using surface plasmon resonance technology we demonstrated the capacity of the poly(A):poly(U) (pA:pU) motif to bind with high affinity to a totally synthetic Tat protein and to inhibit more efficiently the Tat/transactivation response element (TAR) RNA interaction as compared to the poly(I):poly(C) (pI:pC) motif. Furthermore, the pA:pU motif was tenfold more effective in inhibiting Tat‐driven transactivation than the pI:pC motif. Following intranasal immunization of mice, both dsRNA motifs enhanced the antibody (serum and mucosal) and cellular responses to Tat. However, only the serum samples of mice immunized with Tat + pI:pC inhibited Tat‐driven transactivation. The profile of serum antibody subclasses together with the secreted cytokines by Tat‐stimulated splenocyte cultures indicated that both dsRNA motifs favored the induction of a balanced Th1 and Th2 immune response. The demonstration in this study that two dsRNA motifs had a marked effect on Tat/TAR RNA interaction and on the neutralizing capacity of anti‐Tat specific antibody responses highlights their potential for biological applications and the importance of selecting the appropriate motif as an adjuvant for vaccine design.