Jean-Christophe Cintrat

Parthenolide Inhibits Tubulin Carboxypeptidase Activity

Microtubules are centrally involved in cell division, being the principal components of mitotic spindle. Tubulin, the constituent of microtubules, can be cyclically modified on its alpha-subunit by enzymatic removal of the COOH-terminal tyrosine residue by an ill-defined tubulin carboxypeptidase (TCP) and its readdition by tubulin tyrosine ligase (TTL). We and others have previously shown that suppression of TTL and resulting accumulation of detyrosinated tubulin are frequent in human cancers of poor prognosis. Explanations for the involvement of TTL and detyrosinated tubulin in tumor progression arise from the recent discovery that tubulin detyrosination leads to CAP-Gly protein mislocalization, which correlates with defects in spindle positioning during mitosis. Impaired control of spindle positioning is one factor favoring tumor invasiveness. Thus, TCP could be a target for developing novel therapeutic strategies against advanced stages of cancers. Inhibitors of TCP, by reversing abnormal detyrosinated tubulin accumulation in tumor cells, could impair tumor progression. TCP has never been isolated and this has hampered search of specific inhibitors. In this article, we describe a cell-based assay of TCP activity and its use to screen a library of natural extracts for their inhibitory potency. This led to the isolation of two sesquiterpene lactones. We subsequently found that parthenolide, a structurally related compound, can efficiently inhibit TCP. This inhibitory activity is a new specific property of parthenolide independent of its action on the nuclear factor-kappaB pathway. Parthenolide is also known for its anticancer properties. Thus, TCP inhibition could be one of the underlying mechanisms of these anticancer properties.

Squalenoylation Favorably Modifies the in Vivo Pharmacokinetics and Biodistribution of Gemcitabine in Mice

Gemcitabine (2′,2′-difluorodeoxyribofuranosylcytosine; dFdC) is an anticancer nucleoside analog active against wide variety of solid tumors. However, this compound is rapidly inactivated by enzymatic deamination and can also induce drug resistance. To overcome the above drawbacks, we recently designed a new squalenoyl nanomedicine of dFdC [4-(N)-trisnorsqualenoyl-gemcitabine (SQdFdC)] by covalently coupling gemcitabine with the 1,1′,2-trisnorsqualenic acid; the resultant nanomedicine displayed impressively greater anticancer activity compared with the parent drug in an experimental murine model. In the present study, we report that SQdFdC nanoassemblies triggered controlled and prolonged release of dFdC and displayed considerably greater t1/2 (∼3.9-fold), mean residence time (∼7.5-fold) compared with the dFdC administered as a free drug in mice. It was also observed that the linkage of gemcitabine to the 1,1′,2-trisnorsqualenic acid noticeably delayed the metabolism of dFdC into its inactive difluorodeoxyuridine (dFdU) metabolite, compared with dFdC. Additionally, the elimination of SQdFdC nanoassemblies was considerably lower compared with free dFdC, as indicated by lower radioactivity found in urine and kidneys, in accordance with the plasmatic concentrations of dFdU. SQdFdC nanoassemblies also underwent considerably higher distribution to the organs of the reticuloendothelial system, such as spleen and liver (p < 0.05), both after single- or multiple-dose administration schedule. Herein, this paper brings comprehensive pharmacokinetic and biodistribution insights that may explain the previously observed greater efficacy of SQdFdC nanoassemblies against experimental leukemia.

Control of peptide nanotube diameter by chemical modifications of an aromatic residue involved in a single close contact

Supramolecular self-assembly is an attractive pathway for bottom-up synthesis of novel nanomaterials. In particular, this approach allows the spontaneous formation of structures of well-defined shapes and monodisperse characteristic sizes. Because nanotechnology mainly relies on size-dependent physical phenomena, the control of monodispersity is required, but the possibility of tuning the size is also essential. For self-assembling systems, shape, size, and monodispersity are mainly settled by the chemical structure of the building block. Attempts to change the size notably by chemical modification usually end up with the loss of self-assembly. Here, we generated a library of 17 peptides forming nanotubes of monodisperse diameter ranging from 10 to 36 nm. A structural model taking into account close contacts explains how a modification of a few Å of a single aromatic residue induces a fourfold increase in nanotube diameter. The application of such a strategy is demonstrated by the formation of silica nanotubes of various diameters.

N-Methyldihydroquinazolinone Derivatives of Retro-2 with Enhanced Efficacy against Shiga Toxin

The Retro-2 molecule protects cells against Shiga toxins by specifically blocking retrograde transport from early endosomes to the trans-Golgi network. A SAR study has been carried out to identify more potent compounds. Cyclization and modifications of Retro-2 led to a compound with roughly 100-fold improvement of the EC50 against Shiga toxin cytotoxicity measured in a cell protein synthesis assay. We also demonstrated that only one enantiomer of the dihydroquinazolinone reported herein is bioactive.

Syntaxin 5-Dependent Retrograde Transport to the trans-Golgi Network Is Required for Adeno-Associated Virus Transduction

ABSTRACT Intracellular transport of recombinant adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasm, allowing subsequent nuclear import and the initiation of gene expression, remain to be elucidated fully. Others and we previously showed that a significant proportion of viral particles are transported to the Golgi apparatus and that Golgi apparatus disruption caused by the drug brefeldin A efficiently blocks AAV serotype 2 (AAV2) transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of transport to the Golgi apparatus for AAV transduction remains to be established definitively. Here, we show that AAV2 trafficking toward the trans-Golgi network (TGN) and the Golgi apparatus correlates with transduction efficiency and relies on a nonclassical retrograde transport pathway that is independent of the retromer complex, late endosomes, and recycling endosomes. AAV2 transduction is unaffected by the knockdown of syntaxins 6 and 16, which are two major effectors in the retrograde transport of both exogenous and endogenous cargo. On the other hand, inhibition of syntaxin 5 function by small interfering RNA silencing or treatment with cyclized Retro-2 strongly decreases AAV2 transduction and transport to the Golgi apparatus. This inhibition of transduction is observed with several AAV serotypes and a number of primary and immortalized cells. Together, our data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi apparatus is a broadly conserved feature of AAV trafficking that appears to be independent of the identity of the receptors used for viral attachment. IMPORTANCE Gene therapy constitutes a promising approach for the treatment of life-threatening conditions refractory to any other form of remedy. Adeno-associated virus (AAV) vectors are currently being evaluated for the treatment of diseases such as Duchenne muscular dystrophy, hemophilia, heart failure, Parkinsons disease, and others. Despite their promise as gene delivery vehicles, a better understanding of the biology of AAV-based vectors is necessary to improve further their efficacy. AAV vectors must reach the nucleus in order to deliver their genome, and their intracellular transport is not fully understood. Here, we dissect an important step of the intracellular journey of AAV by showing that retrograde transport of capsids to the trans-Golgi network is necessary for gene delivery. We show that the AAV trafficking route differs from that of known Golgi apparatus-targeted cargos, and we raise the possibility that this nonclassical pathway is shared by most AAV variants, regardless of their attachment receptors.

Scalable synthesis of cryptophane-1.1.1 and its functionalization.

Cryptophanes, cage molecules for which xenon exhibits a high affinity, are good candidates for xenon biosensing. Cryptophane-1.1.1 (1) exhibits the highest binding constant for xenon encapsulation in organic solution determined to date. This property suggests that the cryptophane-1.1.1 core (1) is optimal for sensing applications. A high-yielding scalable synthesis of compound 1 is reported as well as an easy way to functionalize it.

(S)-N-Methyldihydroquinazolinones are the Active Enantiomers of Retro-2 Derived Compounds against Toxins

This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of a new compound, named Retro-2.1, active against toxins by inhibiting intracellular trafficking via the retrograde route. The absolute configuration of the bioactive enantiomer has been assigned from X-ray diffraction to the (S)-enantiomer. To date, (S)-Retro-2.1 is the most potent molecule to counteract the cytotoxic potential of ricin and Shiga toxin, with EC50 values of 23 and 54 nM, respectively.

Structural role of counterions adsorbed on self-assembled peptide nanotubes.

Among noncovalent forces, electrostatic ones are the strongest and possess a rather long-range action. For these reasons, charges and counterions play a prominent role in self-assembly processes in water and therefore in many biological systems. However, the complexity of the biological media often hinders a detailed understanding of all the electrostatic-related events. In this context, we have studied the role of charges and counterions in the self-assembly of lanreotide, a cationic octapeptide. This peptide spontaneously forms monodisperse nanotubes (NTs) above a critical concentration when solubilized in pure water. Free from any screening buffer, we assessed the interactions between the different peptide oligomers and counterions in solutions, above and below the critical assembly concentration. Our results provide explanations for the selection of a dimeric building block instead of a monomeric one. Indeed, the apparent charge of the dimers is lower than that of the monomers because of strong chemisorption. This phenomenon has two consequences: (i) the dimer-dimer interaction is less repulsive than the monomer-monomer one and (ii) the lowered charge of the dimeric building block weakens the electrostatic repulsion from the positively charged NT walls. Moreover, additional counterion condensation (physisorption) occurs on the NT wall. We furthermore show that the counterions interacting with the NTs play a structural role as they tune the NTs diameter. We demonstrate by a simple model that counterions adsorption sites located on the inner face of the NT walls are responsible for this size control.

Retrograde Trafficking Inhibitor of Shiga Toxins Reduces Morbidity and Mortality of Mice Infected with Enterohemorrhagic Escherichia coli

ABSTRACT The most deadly outbreak of Escherichia coli O104:H4 occurred in Europe in 2011. Here, we evaluated the effects of the retrograde trafficking inhibitor Retro-2cycl in a murine model of E. coli O104:H4 infection. Systemic treatment with Retro-2cycl significantly reduced body weight loss and improved clinical scores and survival rates for O104:H4-infected mice. The present data established that Retro-2cycl contributes to the protection of mice against O104:H4 infection and may represent a novel approach to limit Shiga toxin-producing Escherichia coli (STEC)-induced toxicity.

Synthesis of [3H2]-(11S,17R)-11,17-Dimethylhentriacontane: a Useful Tool for the Study of the Internalisation of Communication Pheromones of Ant Camponotus vagus

Abstract The convergent synthesis in high enantiomeric and diastereoisomeric purity of [ 3 H 2 ]-(11 S ,17 R )-11,17-dimethylhentriacontane, a communication pheromone of ant Camponotus vagus is described. The stereogenic centres were introduced from commercially available ( S )-citronellol and ( R )-citronellal and the tritiation step was conducted in the last step of the synthesis with tritium gas over the Wilkinson catalyst.