In vitro Evaluation of Isoniazid Derivatives as Potential Agents Against Drug-Resistant Tuberculosis.

The upsurge of multidrug-resistant tuberculosis has toughened the challenge to put an end to this epidemic by 2030. In 2020 the number of deaths attributed to tuberculosis increased as compared to 2019 and newly identified multidrug-resistant tuberculosis cases have been stably close to 3%. Such a context stimulated the search for new and more efficient antitubercular compounds, which culminated in the QSAR-oriented design and synthesis of a series of isoniazid derivatives active against Mycobacterium tuberculosis. From these, some prospective isonicotinoyl hydrazones and isonicotinoyl hydrazides are studied in this work. To evaluate if the chemical derivatizations are generating compounds with a good performance concerning several in vitro assays, their cytotoxicity against human liver HepG2 cells was determined and their ability to bind human serum albumin was thoroughly investigated. For the two new derivatives presented in this study, we also determined their lipophilicity and activity against both the wild type and an isoniazid-resistant strain of Mycobacterium tuberculosis carrying the most prevalent mutation on the katG gene, S315T. All compounds were less cytotoxic than many drugs in clinical use with IC50 values after a 72 h challenge always higher than 25 µM. Additionally, all isoniazid derivatives studied exhibited stronger binding to human serum albumin than isoniazid itself, with dissociation constants in the order of 10-4-10-5 M as opposed to 10-3 M, respectively. This suggests that their transport and half-life in the blood stream are likely improved when compared to the parent compound. Furthermore, our results are a strong indication that the N' = C bond of the hydrazone derivatives of INH tested is essential for their enhanced activity against the mutant strain of M. tuberculosis in comparison to both their reduced counterparts and INH.

Yeast Sphingolipid-Enriched Domains and Membrane Compartments in the Absence of Mannosyldiinositolphosphorylceramide.

The relevance of mannosyldiinositolphosphorylceramide [M(IP)2C] synthesis, the terminal complex sphingolipid class in the yeast Saccharomyces cerevisiae, for the lateral organization of the plasma membrane, and in particular for sphingolipid-enriched gel-like domains, was investigated by fluorescence spectroscopy and microscopy. We also addressed how changing the complex sphingolipid profile in the plasma membrane could influence the membrane compartments (MC) containing either the arginine/ H+ symporter Can1p (MCC) or the proton ATPase Pma1p (MCP). To achieve these goals, wild-type (wt) and ipt1Δ cells, which are unable to synthesize M(IP)2C accumulating mannosylinositolphosphorylceramide (MIPC), were compared. Living cells, isolated plasma membrane and giant unilamellar vesicles reconstituted from plasma membrane lipids were labelled with various fluorescent membrane probes that report the presence and organization of distinct lipid domains, global order, and dielectric properties. Can1p and Pma1p were tagged with GFP and mRFP, respectively, in both yeast strains, to evaluate their lateral organization using confocal fluorescence intensity and fluorescence lifetime imaging. The results show that IPT1 deletion strongly affects the rigidity of gel-like domains but not their relative abundance, whereas no significant alterations could be perceived in ergosterolenriched domains. Moreover, in these cells lacking M(IP)2C, a clear alteration in Pma1p membrane distribution, but no significant changes in Can1p distribution, were observed. Thus, this work reinforces the notion that sphingolipid-enriched domains distinct from ergosterol-enriched regions are present in the S. cerevisiae plasma membrane and suggests that M(IP)2C is important for a proper hydrophobic chain packing of sphingolipids in the gel-like domains of wt cells. Furthermore, our results strongly support the involvement of sphingolipid domains in the formation and stability of the MCP, possibly being enriched in this compartment.

Sphingolipid hydroxylation in mammals, yeast and plants - An integrated view.

This review is focused on sphingolipid backbone hydroxylation, a small but widespread structural feature with profound impact on membrane biophysical properties. We start by summarizing sphingolipid metabolism in mammalian cells, yeast and plants, focusing on how distinct hydroxylation patterns emerge in different eukaryotic kingdoms. Then, a comparison of the biophysical properties in membrane model systems and cellular membranes from diverse organisms is made. From an integrative perspective, these results can be rationalized considering that superficial hydroxyl groups in the backbone of sphingolipids (by intervening in the H-bond network) alter the balance of favorable interactions between membrane lipids. They may strengthen the bonding or compete with other hydroxyl groups, in particular the one of membrane sterols. Different sphingolipid hydroxylation patterns can stabilize/disrupt specific membrane domains or change whole plasma membrane properties, and therefore be important in the control of protein distribution, function and lateral diffusion and in the formation and overtime stability of signaling platforms. The recent examples explored throughout this review unveil a potentially key role for sphingolipid backbone hydroxylation in both physiological and pathological situations, as it can be of extreme importance for the proper organization of cell membranes in mammalian cells, yeast and, most likely, also in plants.