Biological membranes are fundamental components of all living cells. Their biophysical properties are critical for their numerous functions of mammalian cells including traffic, anchorage of receptors and cell signalling. In this respect, the existence of clusters of proteins and lipids appears as a critical biophysical property of lipid membranes. One of the most often considered models is the raft hypothesis i.e. the partition of lipids between liquid disordered and ordered phases, the latter being enriched in sphingolipids and cholesterol. In prokaryotes also, the existence of lipid domains is now widely accepted with the three main lipids found in bacterial membranes (phosphatidylethanolamine, phosphatidylglycerol and cardiolipin) organized in microdomains.
Our work, focused on the interactions between drugs and lipid membranes, is persued with the aim to provide a more comprehensive and biologically relevant picture of the drug membrane interaction and how the effect of these interactions can modify the biophysical properties of the membranes. Results are put in relation with anticancer and antibacterial activities.
The main questions we address are related to (i) the type of interactions between drugs and lipids, (ii) the modifications of membrane biophysical properties induced by drugs, and (iii) the consequences of these modifications on cellular pharmacokinetics,activity, or toxicity of drugs. Most of these studies are performed by using membrane models (supported bilayers, liposomes[SUVs, LUVs; GUVs]) mimicking (i) eukaryotic and (ii) bacterial membranes.
In close collaboration, we developed a range of complementary methods including AFM, 31PNMR, ellipsometry, dynamic light scattering, fluorescence spectroscopy (Laurdan, DPH, DHE, calcein, octadecylrhodamine B…) and confocal microscopy.
(i) Over the three last years, we investigated the interaction between lipids of eukaryotic cells and amphiphilic drugs and peptides.
The aim of this work performed on eukaryotic cells was to investigate the membrane effects induced by amphiphilic pharmacological compounds in relation with their pharmaceutical interests. We focused on saponins (α-hederin) and lipopeptides (surfactin) both suggested as potential anticancer drugs.
By investigating the molecular mechanism involved in necrosis and apoptosis in leukemic monocytes induced by α-hederin, a monodesmosidic triterpenoid saponin, we demonstrated the critical role of cholesterol.
On models of membranes, we showed that α-hederin induced membrane permeabilization by a mechanism which is mainly driven by the formation of
saponin/cholesterol domains and the induction of membrane curvature. The latter was dependent on the sugar chain branched on C3 of the aglycone, hederagenin. α-hederin induced phase separation which would be stabilized by a coupling between local composition and monolayer curvature.
The lipid phase separation can be observed without any permeabilization of the membrane. Together these results (Figure 1) could be related to the capacity of α-hederin to induce apoptosis in cells and to its potential anticancer effect.
Figure 1 : Lorent et al, Langmuir, 2014
Regarding surfactin, a bacterial cyclic amphiphilic lipopeptide known to selectively kill cancer cells, we showed that the presence of rigid domains can play an essential role in the first step of its insertion within the lipid bilayer and its interaction with both the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes has been proposed (Figure 2). At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes.
At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased the order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.
Figure 2. Monolayer grid of 200X200 lipids calculated by the Big Monolayer Method. Each pixel represents a molecule.
Blue: DPPC molecule; yellow: DOPC molecule; green: surfactin Molecule. (A) DOPC: DPPC at 1:1 molar ratio, (B)
DOPC:DPPC:surfactin at 1:1:0.1 molar ratio, (C)
DOPC:DPPC/surfactin at 1:1:0.3 molar ratio (Deleu, Lorent et al, Biochem. Biophys. Acta, 2013).
Taking benefit from our expertise, we extended our approaches to ginsenosides and to Budesonide/Cyclodextrins complexes with the aim to understand how
changes of lipid phases could be related to different activities on cell membranes and to pharmacological effects.
(ii) In parallel to studies performed on lipids found in eukaryotic cells, we are also interested in the study of the mode of action of new antibiotics acting on bacterial lipid membrane.
The increased bacterial drug-resistance against traditional antibiotics becomes a major challenge in healthcare and creates an urgent need to develop novelcompounds to treat infectious diseases. Taking benefit from a collaboration with chemists working the Département of Pharmacochimie (the University of Grenoble, France), we explored the interactions of original amphiphilic derivatives of the aminoglycoside neamine on lipids found in membranes of Gram-negative bacteria.
We showed that some derivatives of neamine are active against sensitive and resistant P. aeruginosa strains as well as S. aureus strains including strains expressing enzymes modifying aminoglycosides, efflux pumps, or rRNA methylases. The mechanism of action is different from inhibition of protein synthesis as observed for conventional aminoglycosides, and results from membrane destabilization.
To decipher, at the molecular level, the mechanism involved in this antimicrobial effect, we determined how the loss of membrane integrity due to LPS binding can affect the integrity of the cytoplasmic leaflet of the outer membrane as well as the integrity of the inner membrane. On P. aeruginosa, we monitored membrane permeabilization (NPN and PI assays) and membrane depolarization (DiSC3(5) fluorescence). The interactions of selected amphiphilic aminoglycosides with the three main lipids of the inner membrane of P. aeruginosa, PE (phosphatidylethanolamine), PG (phosphatidylglycerol) and CL (cardiolipin) and their effects, is investigated by using relevant membrane models, GUVs (Giant Unilamellar vesicles) for confocal microscopy and lipid monolayers for Langmuir isotherm compression. By confocal microscopy, we are monitoring the lipid domains especially those enriched in cardiolipin.
To enhance the selectivity, we are going to investigate a series of new derivatives varying by the nature of the hydrophobic tail (naphtyl, alkyl, alkyl) as well as the central backbone (neamine versus neosamine) or the position and the number of substitution on the central backbone to define optimal amphiphilicity (Figure 3). This should offer promising prospects in the search for new antibacterials against drug – or biocide – resistant strains.
Figure 3: Zimmerman et al, J. Med. Chem. 2013
Our results bring into light fundamental concepts which could be important in membrane-lipid therapy in which the molecular targets are the lipids and the structure they form. The role of lipids can be (i) to facilitate membrane bending and the formation of highly curved intermediates, reducing the energy barriers of fission and fusion and (ii) to recruit specialized proteins. Influencing curvature directly as well as indirectly by targeting negative intrinsic curvature of lipids or in impairing the soft mechanical behavior could be a new approach for antibiotic design.