Elucidation of a mechanism of cell lysis by chorhexidine : a biophysical approach

Chlorhexidine is an effective antiseptic used widely in disinfecting products (hand soap), oral products (mouthwash), and is known to have potential applications in the textile industry. Chlorhexidine has been studied extensively through a biological and biochemical lens, showing evidence that it attacks the semipermeable membrane in bacterial cells. Although extremely lethal to bacterial cells, the present understanding of the exact mode of action of chlorhexidine is incomplete. A biophysical approach has been taken to investigate the potential location of chlorhexidine in the lipid bilayer. Deuterium nuclear magnetic resonance was used to characterize the molecular arrangement of mixed phospholipid/drug formulations. Powder spectra were analyzed using the de-Pake-ing technique, a method capable of extracting both the orientation distribution and the anisotropy distribution functions simultaneously. The results from samples of protonated phospholipids mixed with deuterium-labelled chlorhexidine are compared to those from samples of deuterated phospholipids and protonated chlorhexidine to determine its location in the lipid bilayer. A series of neutron scattering experiments were also conducted to study the biophysical interaction of chlorhexidine with a model phospholipid membrane of DMPC, a common saturated lipid found in bacterial cell membranes. The results found the hexamethylene linker to be located at the depth of the glycerol/phosphate region of the lipid bilayer. As drug concentration was increased in samples, a dramatic decrease in bilayer thickness was observed. Differential scanning calorimetry experiments have revealed a depression of the DMPC bilayer gel-to-lamellar phase transition temperature with an increasing drug concentration. The enthalpy of the transition remained the same for all drug concentrations, indicating a strictly drug/headgroup interaction, thus supporting the proposed location of chlorhexidine. In combination, these results lead to the hypothesis that the drug is folded approximately in half on its hexamethylene linker, with the hydrophobic linker at the depth of the glycerol/phosphate region of the lipid bilayer and the hydrophilic chlorophenyl groups located at the lipid headgroup. This arrangement seems to suggest that the drug molecule acts as a wedge to disrupt the bilayer. In vivo, this should make the cell membrane leaky, which is in agreement with a wide range of bacteriological observations.

The Synthesis of Imidazole Fatty Acid Conjugates as Inhibitors of Apoptosis

Ionizing radiation is known to initiate apoptosis in mammalian cells by causing the transformation of cytochrome c into a peroxidase, which results in the specific peroxidation of the mitochondrial phospholipid cardiolipin. Here we report the design and synthesis of 8 imidazole fatty acid derivatives that bind to the cyt c:CL complex and inhibit the peroxidase activity required for the initiation of apoptosis. We postulate that imidazole acts as a sixth ligand to the haem iron and stops the interaction with H2O2. Two mitochondrially directed analogues (3-hydroxypropyl)triphenylphosphonium esters) of 12-imidazole-stearic acid and 12-imidazole-oleic acid not only were demonstrated to be peroxidase inhibitors in vitro, but were also extraordinarily effective in protecting mice from lethal doses (9 Gy) of ionization radiation. We studied the structure activity relationship to a group of triphenyl phosphonium derivatives containing imidazole at different positions on the fatty acid chain, and observed that the C8-imidazole stearate analogue had marginally better activity than the others. But overall, the structure activity result were remarkable “flat” with all compounds prepared having rather similar inhibitory strength. We also synthesized carnitine mono and di-esters of 12-imidazole fatty acids but full biological data is not yet available for these compounds.

The Role of Membrane Lipid Composition on Skeletal Muscle Damage in the Rodent Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy is a X-linked muscle disease, which leads to alterations in membrane phospholipid fatty acid (FA) composition and skeletal muscle damage. Increased membrane saturated FA in muscular dystrophy may suggest its association with increased susceptibility (as being the cause or consequence) to muscle damage. It was hypothesised that increased saturation is positively correlated to increased muscle damage. Correlations were hypothesized to be greater in extensor digitorum longus (EDL) at 20 weeks compared to soleus (SOL) at 10 weeks in dystrophin deficient (mdx) mice. Increased saturation was correlated to damage in EDL at both 10 and 20 weeks, with stronger correlations at 10 weeks. The results suggest that membrane PL FA composition may be associated with damage through two possible means. Increased saturation may be a cause or consequence of membrane damage. Association of membrane composition with eccentric induced damage has underscored the importance of saturated PL FA compositions in damage to dystrophic myofibres.

The Ligand and Membrane-binding Behaviour of the Phosphatidylinositol Transfer Proteins (PITPa & PITPb)

Human Class I phosphatidylinositol transfer proteins (PITPs) exists in two forms: PITPα and PITPβ. PITPs are believed to be lipid transfer proteins based on their capacity to transfer either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane compartments in vitro. In Drosophila, the PITP domain is found to be part of a multi-domain protein named retinal degeneration B (RdgBα). The PITP domain of RdgBα shares 40 % sequence identity with PITPα and has been shown to possess PI and PC binding and transfer activity. The detailed molecular mechanism of ligand transfer by the human PITPs and the Drosophila PITP domain remains to be fully established. Here, we investigated the membrane interactions of these proteins using dual polarization interferometry (DPI). DPI is a technique that measures protein binding affinity to a flat immobilized lipid bilayer. In addition, we also measured how quickly these proteins transfer their ligands to lipid vesicles using a fluorescence resonance energy transfer (FRET)-based assay. DPI investigations suggest that PITPβ had a two-fold higher affinity for membranes compared to PITPα. This was reflected by a four-fold faster ligand transfer rate for PITPβ in comparison to PITPα as determined by the FRET assay. Interestingly, DPI analysis also demonstrated that PI-bound human PITPs have lower membrane affinity compared to PC-bound PITPs. In addition, the FRET studies demonstrated the significance of membrane curvature in the ligand transfer rate of PITPs. The ligand transfer rate was higher when the accepting vesicles were highly curved. Furthermore, when the accepting vesicles contained phosphatidic acid (PA) which have smaller head groups, the transfer rate increased. In contrast, when the accepting vesicles contained phosphoinositides which have larger head groups, the transfer rate was diminished. However, PI, the favorite ligand of PITPs, or the presence of anionic lipids did not appear to influence the ligand transfer rate of PITPs. Both DPI and FRET examinations revealed that the PITP domain of RdgBα was able to bind to membranes. However, the RdgBα PITP domain appears to be a poor binder and transporter of PC.