Laurence Clary

Synthesis of single- and double-chain fluorocarbon and hydrocarbon galactosyl amphiphiles and their anti-HIV-1 activity

Galactosylceramide (GalCer) is an alternative receptor allowing HIV-1 entry into CD4(-)/GalCer(+) cells. This glycosphingolipid recognizes the V3 loop of HIV gp120, which plays a key role in the fusion of the HIV envelope and cellular membrane. To inhibit HIV uptake and infection, we designed and synthesized analogs of GalCer. These amphiphiles and bolaamphiphiles consist of single and double hydrocarbon and/or fluorocarbon chain beta-linked to galactose and galactosamine. They derive from serine (GalSer), cysteine (GalCys), and ethanolamine (GalAE). The anti-HIV activity and cytotoxicity of these galactolipids were evaluated in vitro on CEM-SS (a CD4(+) cell line), HT-29, a CD4(-) cell line expressing high levels of GalCer receptor, and/or HT29 genetically modified to express CD4. GalSer and GalAE derivatives, tested in aqueous medium or as part of liposome preparation, showed moderate anti-HIV-1 activities (IC50 in the 20-220 microM range), whereas none of the GalCys derivatives was found to be active. Moreover, only some of these anti-HIV active analogs inhibited the binding of [3H]suramin (a polysulfonyl compound which displays a high affinity for the V3 loop) to SPC3, a synthetic peptide which contains the conserved GPGRAF region of the V3 loop. Our results most likely indicate that the neutralization of the virion through masking of this conserved V3 loop region is not the only mechanism involved in the HIV-1 antiviral activity of our GalCer analogs.

Membrane permeability and stability of liposomes made from highly fluorinated double-chain phosphocholines derived from diaminopropanol, serine or ethanolamine

The release of encapsulated carboxyfluorescein (CF) from liposomes made from various fluorinated amido-connected double-chain phosphocholines and their membrane permeability have been investigated at 37 degrees C in buffer and in human serum. These fluorinated membranes and liposomes display lower permeability coefficients and are able to retain more efficiently encapsulated CF than any of their respective conventional counterparts. Several of these liposomes are as effective as the first generation of liposomes based on fluorinated phosphatidylcholines, indicating that the chemical junction (ester/amide) and nature of the unit (glycerol, diaminopropanol, serine, ethanolamine) connecting the hydrophobic chains to the phosphocholine polar head have no significant effect on permeability and CF release. Our results show further that a fluorinated intramembrane layer reduces significantly the permeability of membranes in a liquid-crystalline state, protects the liposomes from the destabilizing effects of serum, and even increases their stability (in terms of dye retention) in serum when the membranes are in the gel state.

Synthesis and evaluation of the in vivo tolerance of amido fiuorocarbon/fluorocarbon and fluorocarbon/hydrocarbon double-chain phosphocholines deriving from diaminopropanols and serine

Abstract The syntheses of various fluorocarbon/fluorocarbon and fluorocarbon/hydrocarbon amido-connected phosphocholines derived from diaminopropanols and serine are described. They were best obtained by phosphorylation of suitable alcohol precursors using 2-chloro-2-oxo-1,3,2-dioxaphospholane and subsequent ring opening with trimethylamine. The di-alkylamidopropanols were prepared by acylation, using perfluoroalkylated acid chlorides, of 1,3-diamino-2-propanol or of 2,3-diamino-1-propionic methylester followed by reduction of the ester bond. The perfluoroalkylated N-alkanoyl-serine alkylamides were prepared by condensation of Boc-O-Bn-L-serine with an aliphatic or perfluoroalkylated amine, Boc-deprotection, acylation and then hydrogenolysis for benzyldeprotection. Acute toxicity evaluations indicate a very promising in vivo tolerance for these series of amido-linked compounds.

Phase behavior of fluorocarbon and hydrocarbon double-chain hydroxylated and galactosylated amphiphiles and bolaamphiphiles. Long-term shelf-stability of their liposomes.

This paper describes the morphological characterization, by freeze-fracture electron microscopy, and the thermotropic phase behavior, by differential scanning calorimetry and/or X-ray scattering, of aqueous dispersions of various hydroxylated and galactosylated double-chain amphiphiles and bolaamphiphiles, several of them containing one or two hydrophobic fluorocarbon chains. Colloidal systems are observed in water with the hydroxylated hydrocarbon or fluorocarbon bolaamphiphiles only when they are dispersed with a co-amphiphile such as rac-1,2-dimyristoylphosphatidylcholine (DMPC) or rac-1,2-distearoylphosphatidylcholine (DSPC). Liposomes are formed providing the relative content of bolaamphiphiles does not exceed 20% mol. Most of these liposomes can be thermally sterilized and stored at room temperature for several months without any significant modification of their size and size distribution. The hydrocarbon galactosylated bolaamphiphile HO[C24][C12]Gal forms in water a lamellar phase (the gel to liquid-crystal phase transition is complete at 45 degrees C) and a Im3m cubic phase above 47 degrees C. The fluorocarbon HO[C24][F6C5]Gal analog displays a more complex and metastable phase behavior. The fluorinated non-bolaform galactosylated [F8C7][C16]AEGal and SerGal amphiphiles form lamellar phases in water. Low amounts (10% molar ratio) of the HO[C24][F6C5]Gal or HO[C24][C12]Gal bolaamphiphiles or of the single-headed [F8C7][C16]AEGal improve substantially the shelf-stability of reference phospholipon/cholesterol 2/1 liposomes. These liposomes when co-formulated with a single-headed amphiphile from the SerGal series are by far less stable.

synthesis of single- and double-chain fluorocarbon and hydrocarbon β-linked galactose amphiphiles derived from serine

Abstract Single- and double-chain β-linked galactose amphiphiles derived from serine were synthesized. Both types of compounds have potential as material for the formulation of liposomal drug carrier and targeting systems and as HIV inhibitors.

Polymorphic phase behavior of fluorocarbon double-chain phosphocholines derived from diaminopropanol, serine and ethanolamine and long-term shelf stability of their liposomes

Abstract This paper describes the morphological characterization, by freeze-fracture electron microscopy, and the thermotropic phase behavior, by differential scanning calorimetry, of aqueous dispersions of various fluorocarbon/fluorocarbon or mixed fluorocarbon/hydrocarbon double-chain 2,3- or 1,3-diamidopropano-, diamidoserino- or amidoethyl-phosphocholines (PC). These fluorinated PCs form lamellar phases and liposomes, as their ester or ether glycerol-based analogs. The effects on the thermodynamic parameters associated with these lamellar phases of several structural elements — chemical junction (secondary CONHR or tertiary CONRR), connecting unit (diaminopropanol, serine or ethanolamine), number and length of the perfluoroalkylated chains, length of the fluorinated tail and hydrocarbon — are discussed. These data are compared with those reported for fluorocarbon or hydrocarbon glycerophosphocholines and hydrocarbon diamidophosphocholines, and for sphingomyelins. Most of the liposomes formed from the fluorinated amido-connected PCs display a remarkable long-term shelf stability: they can be thermally sterilized and stored at room temperature for several months without any significant modification of their size and size distribution.

Transmembrane pH-driven Na+ permeability of fluorinated phospholipid-based membranes

The permeability to the H+/Na+ exchange of fluorinated phospholipid-based membranes has been evaluated by measuring the dissipation rate of a liposomal transmembrane pH gradient in the presence of Na+. The fluorinated liposomes are made from fluorocarbon/hydrocarbon or fluorocarbon/fluorocarbon double-chain ether-connected glycerophosphocholines or amido-connected phosphocholines deriving from diaminopropanol or serine. The fluorocarbon/hydrocarbon mixed-chain phospholipids, as compared to the fluorocarbon/fluorocarbon ones, form membranes that are substantially more able to maintain a transmembrane pH gradient in the presence of NA+ and display a lower Na+ permeability. However, these membranes are more permeable to the H+/Na/ exchange than conventional DSPC (1,2-distearoylphosphatidylcholine) ones. Our results indicate a detrimental impact of the membrane fluorination degree on H+/Na+ permeability: the lower the fluorination degree of the membrane, the lower its H+/Na+ permeability. Concerning structure/permeability relationships, it appears that the replacement of the ester connecting bond in their fluorinated phosphatidylcholine analogues for an ether or amide one lowers the transmembrane H+/Na+ exchange.

Molecular order and mobility within membranes made from fluorocarbon/fluorocarbon and fluorocarbon/hydrocarbon double-chain ether and amide phospholipids

Abstract The order of membranes formed by various ether- or amide-connected fluorocarbon/fluorocarbon or mixed fluorocarbon/hydrocarbon double-chain phosphocholines which derive, respectively, from glycerol or diaminopropanol or serine, has been investigated using fluorescence anisotropy of TMA-DPH, and compared to that of membranes made from fluorinated or conventional ester-connected phosphatidylcholines. In the gel phase, membrane order is almost not affected by the molecular structure of the phospholipid. By contrast, in the liquid-crystalline state, it decreases on going (i) from membranes made from the fluorocarbon/fluorocarbon phospholipids to those formed by mixed fluorocarbon/hydrocarbon phospholipids, then to conventional ones, (the rigidifying effects of the fluorinated tails, their number and length is thus becoming noticeable) or (ii) from membranes made from fluorinated diamidopropano- or diamidoserinophosphocholines to those made from ester- or ether-connected glycerophosphocholines. The ester- versus ether-connection or 1,2- versus 1,3-connection of the chains on glycerol have no detectable impact on membrane order.

New Caspase-1 inhibitor by scaffold hopping into bio-inspired 3D-fragment space

Virtual fragmentation of a library of 12,000 compounds inspired by natural products led to a dataset of 153,000 fragments that was used as a source to identify effective P2-P3 scaffold replacement solutions for peptidic Caspase-1 inhibitors. Our strategy led to the identification of an original 2-azabicyclo-octane scaffold (2-ABO) that was further elaborated into the potent Caspase-1 inhibitor CD10847 (IC50 = 17 nM). The crystal structure of Caspase-1 in complex with CD10847 was obtained, and its binding mode was shown to be similar to the one predicted by docking and in good agreement with other known inhibitors.