Sex-Specific Patterns of Diaphragm Phospholipid Content and Remodeling during Aging and in a Model of SELENON-Related Myopathy.

Growing evidence shows that the lipid bilayer is a key site for membrane interactions and signal transduction. Surprisingly, phospholipids have not been widely studied in skeletal muscles, although mutations in genes involved in their biosynthesis have been associated with muscular diseases. Using mass spectrometry, we performed a phospholipidomic profiling in the diaphragm of male and female, young and aged, wild type and SelenoN knock-out mice, the murine model of an early-onset inherited myopathy with severe diaphragmatic dysfunction. We identified 191 phospholipid (PL) species and revealed an important sexual dimorphism in PLs in the diaphragm, with almost 60% of them being significantly different between male and female animals. In addition, 40% of phospholipids presented significant age-related differences. Interestingly, SELENON protein absence was responsible for remodeling of 10% PL content, completely different in males and in females. Expression of genes encoding enzymes involved in PL remodeling was higher in males compared to females. These results establish the diaphragm PL map and highlight an important PL remodeling pattern depending on sex, aging and partly on genotype. These differences in PL profile may contribute to the identification of biomarkers associated with muscular diseases and muscle aging.

Hacd2 deficiency in mice leads to an early and lethal mitochondrial disease.

OBJECTIVE: Mitochondria fuel most animal cells with ATP, ensuring proper energetic metabolism of organs. Early and extensive mitochondrial dysfunction often leads to severe disorders through multiorgan failure. Hacd2 gene encodes an enzyme involved in very long chain fatty acid (C ≥ 18) synthesis, yet its roles in vivo remain poorly understood. Since mitochondria function relies on specific properties of their membranes conferred by a particular phospholipid composition, we investigated if Hacd2 gene participates to mitochondrial integrity. METHODS: We generated two mouse models, the first one leading to a partial knockdown of Hacd2 expression and the second one, to a complete knockout of Hacd2 expression. We performed an in-depth analysis of the associated phenotypes, from whole organism to molecular scale. RESULTS: Thanks to these models, we show that Hacd2 displays an early and broad expression, and that its deficiency in mice is lethal. Specifically, partial knockdown of Hacd2 expression leads to death within one to four weeks after birth, from a sudden growth arrest followed by cachexia and lethargy. The total knockout of Hacd2 is even more severe, characterized by embryonic lethality around E9.5 following developmental arrest and pronounced cardiovascular malformations. In-depth mechanistic analysis revealed that Hacd2 deficiency causes altered mitochondrial efficiency and ultrastructure, as well as accumulation of oxidized cardiolipin. CONCLUSIONS: Altogether, these data indicate that the Hacd2 gene is essential for energetic metabolism during embryonic and postnatal development, acting through the control of proper mitochondrial organization and function.

Influence of Hydrophobic Groups Attached to Amphipathic Polymers on the Solubilization of Membrane Proteins along with Their Lipids.

One of the biggest challenges in membrane protein (MP) research is to secure physiologically relevant structural and functional information after extracting MPs from their native membrane. Amphipathic polymers represent attractive alternatives to detergents for stabilizing MPs in aqueous solutions. The predominant polymers used in MP biochemistry and biophysics are amphipols (APols), one class of which, styrene maleic acid (SMA) copolymers and their derivatives, has proven particularly efficient at MP extraction. In order to examine the relationship between the chemical structure of the polymers and their ability to extract MPs from membranes, we have developed two novel classes of APols bearing either cycloalkane or aryl (aromatic) rings, named CyclAPols and ArylAPols, respectively. The effect on solubilization of such parameters as the density of hydrophobic groups, the number of carbon atoms and their arrangement in the hydrophobic moieties, as well as the charge density of the polymers was evaluated. The membrane-solubilizing efficiency of the SMAs, CyclAPols, and ArylAPols was compared using as models (i) two MPs, BmrA and a GFP-fused version of LacY, overexpressed in the inner membrane of Escherichia coli, and (ii) bacteriorhodopsin, naturally expressed in the purple membrane of Halobacterium salinarum. This analysis shows that, as compared to SMAs, the novel APols feature an improved efficiency at extracting MPs while preserving native protein-lipid interactions.

Unequal Behaviour between Hydrolysable Functions of Nirmatrelvir under Stress Conditions: Structural and Theoretical Approaches in Support of Preformulation Studies.

Nirmatrelvir is an antiviral drug approved for the treatment of COVID-19. The available dosage form consists of tablets marketed under the brand name PAXLOVID®. Although knowledge of nirmatrelvir's intrinsic stability may be useful for any potential development of other pharmaceutical forms, no data regarding this matter is available to date. Preliminary forced degradation studies have shown that the molecule is stable under oxidative and photolytic conditions, while hydrolytic conditions, both acidic and basic, have proven deleterious. Indeed, the molecule presents a priori several functions that can undergo hydrolysis, i.e., three amide moieties and a nitrile function. However, considering the degradation products formed under forced conditions and which were detected and identified by LC-UV-HRMSn, the hydrolysis process leading to their formation is selective since it involved only 2 of the 4 hydrolysable functions of the molecule. Ab initio studies based on density functional theory (DFT) have helped better understand these reactivity differences in aqueous media. Some hydrolyzable functions of nirmatrelvir differ from others in terms of electrostatic potential and Fukui functions, and this seems to correlate with the forced degradation outcomes.

Design, synthesis and biological evaluation of quinoline-2-carbonitrile-based hydroxamic acids as dual tubulin polymerization and histone deacetylases inhibitors.

A series of quinoline and quinazoline analogs were designed and synthesized as new tubulin polymerization (TP) and histone deacetylases (HDAC) inhibitors. Compounds 12a and 12d showed the best cytotoxicity activities against a panel of human cancer cell lines with an averaged IC50 value of 0.6 and 0.7 nM, respectively. Furthermore, these lead compounds showed good activities against CA-4-resistant colon-carcinoma and multidrug-resistant leukemia cells. In addition, compounds 12a and 12d induced HT29 cell cycle arrest in the G2/M phase and produced caspase-induced apoptosis of HT29 cells through mitochondrial dysfunction. Also, 12a and 12d inhibited HDAC8, 6, and 11 activities. Furthermore, lead compound 12a exhibited higher metabolic stability than isoCA-4 and was highly potent in suppressing tumor growth in the fibrosarcoma MCA205 tumor model. Collectively, these studies suggest that 12a represents a new dual inhibitor of TP and HDAC activities, which makes it a suitable candidate for further investigations in clinical development.

Rapid assessment of fatty acyls chains of phospholipids and plasmalogens by atmospheric pressure chemical ionization in positive mode and high-resolution mass spectrometry using in-source generated monoacylglycerol like fragments intensities.

We recently published a new concept using monoacylglycerol-like fragments [MG+H-H2O]+ (ions B) produced in-source by atmospheric pressure photoionization in positive mode and high-resolution mass spectrometry for the determination of the fatty acyl (FA) composition of triacylglycerols (TGs) from plant oils. This study extends the concept to the phospholipids (PLs) category and shows that the APCI+ source can also be used. Moreover, the coupling with NP-LC allows to simultaneously analyze different PLs classes in the same sample. We compared the relative intensities of the ions B produced in-source to the % composition of FAs determined by GC-FID. In the case of PLs from natural extracts composed exclusively of diacyl-PLs, the relative intensities of ions B are close to the % of the FAs obtained by GC-FID. This approach is not directly useable for extracts containing plasmalogens (P-PLs). For these PLs, acidic hydrolysis by HCl fumes allows hydrolyzing selectively vinyl ether functions to form lyso-PLs. The analysis of hydrolyzed extracts makes it possible to obtain the composition of P-PLs FAs thanks to the lyso-PLs thus formed, while the diacyl-PLs composition remains unchanged. Unlike GC-FID FAs determination, this approach allows a distinction between the diacyl-PLs and P-PLs FAs composition. We also found that the ion B intensities were consistent among the PL classes (PG, PE, PA, PI, CL, PS and PC) and lyso- forms (LPE and LPC). In the case of the diacyl-PLs extracts analyzed, no statistically significant differences were found between the PLs FAs compositions calculated from ion B intensities and the corresponding GC-FID data. A weighting coefficient was applied to correct ion B intensities issued from polyunsaturated FAs with three or more double bonds. The fatty alkenyls composition of P-PLs could also be calculated from the % intensities of specific ions.

Gemcitabine lipid prodrug nanoparticles: Switching the lipid moiety and changing the fate in the bloodstream.

A simple approach to achieve a lipoprotein (LP)-mediated drug delivery is to trigger the spontaneous drug insertion into endogenous lipoproteins in the bloodstream, by means of its chemical modification. Nanoparticles (NPs) made of the squalene-gemcitabine (SQGem) conjugate were found to have a high affinity for plasma lipoproteins while free gemcitabine did not, suggesting a key role of the lipid moiety in this event. Whether the drug conjugation to cholesterol, one of the major lipoprotein-transported lipids, could also promote an analogous interaction was a matter of question. NPs made of the cholesterol-gemcitabine conjugate (CholGem) have been herein thoroughly investigated for their blood distribution profile both in vitro and in vivo. Unexpectedly, contrarily to SQGem, no trace of the CholGem prodrug could be found in the lipoprotein fractions, nor was it interacting with albumin. The investigation of isolated NPs and NPs/LPs physical mixtures provided a further insight into the lack of interaction of CholGem NPs with LPs. Although essential for allowing the self-assembly of the prodrug into nanoparticles, the lipid moiety may not be sufficient to elicit interaction of the conjugated drug with plasma lipoproteins but the whole NP physicochemical features must be carefully considered.

Phospholipids: Identification and Implication in Muscle Pathophysiology.

Phospholipids (PLs) are amphiphilic molecules that were essential for life to become cellular. PLs have not only a key role in compartmentation as they are the main components of membrane, but they are also involved in cell signaling, cell metabolism, and even cell pathophysiology. Considered for a long time to simply be structural elements of membranes, phospholipids are increasingly being viewed as sensors of their environment and regulators of many metabolic processes. After presenting their main characteristics, we expose the increasing methods of PL detection and identification that help to understand their key role in life processes. Interest and importance of PL homeostasis is growing as pathogenic variants in genes involved in PL biosynthesis and/or remodeling are linked to human diseases. We here review diseases that involve deregulation of PL homeostasis and present a predominantly muscular phenotype.

Anticancer properties of indole derivatives as IsoCombretastatin A-4 analogues.

In this study, a variety of original ligands related to Combretastatin A-4 and isoCombretastatin A-4, able to inhibit the tubulin polymerization into microtubules, was designed, synthesized, and evaluated. Our lead compound 15d having a quinazoline as A-ring and a 2-substituted indole as B-ring separated by a N-methyl linker displayed a remarkable sub-nanomolar level of cytotoxicity (IC50 < 1 nM) against 9 human cancer cell lines.

Cochleate formulations of Amphotericin b designed for oral administration using a naturally occurring phospholipid.

The purpose of this work was to formulate the poor soluble antifungal and antiparasitic agent Amphotericin B (AmB) in cost-effective lipid-based formulations suitable for oral use in developing countries, overcoming the limitations of poor water solubility, nephrotoxicity and low oral bioavailability. The antifungal agent was formulated, at different molar proportions, in cochleate nanocarriers prepared using an accessible naturally occurring phospholipid rich in phosphatidylserine (Lipoid PSP70). These nanoassemblies were prepared by condensation of negatively charged phospholipid membrane vesicles with divalent cations (Ca2+). Small-angle X-ray scattering studies revealed the Ca2+-triggered condensation of loosely packed multilamellar vesicles into tightly packed bilayers of strongly dehydrated multilamellar organization characterized by narrow Bragg peaks. Transmission electron microscopy and quasi-elastic light scattering studies demonstrated the formation of nanosized particles. AmB drug loading was above 55% in all formulations. Circular dichroism demonstrated the prevalence of monomeric and complexed forms of AmB over toxic aggregates. The stability of AmB in gastric medium was improved by loading in cochleates and its release in gastrointestinal media was retarded. Confocal microscopy studies revealed the in-vitro interactions of Lipoid PSP70-based cochleates with Caco2 intestinal cell monolayers. The results suggest that the low-cost AmB-loaded cochleates may increase the therapeutic range of this drug.