Guiding the choice of informatics software and tools for lipidomics research applications.

Progress in mass spectrometry lipidomics has led to a rapid proliferation of studies across biology and biomedicine. These generate extremely large raw datasets requiring sophisticated solutions to support automated data processing. To address this, numerous software tools have been developed and tailored for specific tasks. However, for researchers, deciding which approach best suits their application relies on ad hoc testing, which is inefficient and time consuming. Here we first review the data processing pipeline, summarizing the scope of available tools. Next, to support researchers, LIPID MAPS provides an interactive online portal listing open-access tools with a graphical user interface. This guides users towards appropriate solutions within major areas in data processing, including (1) lipid-oriented databases, (2) mass spectrometry data repositories, (3) analysis of targeted lipidomics datasets, (4) lipid identification and (5) quantification from untargeted lipidomics datasets, (6) statistical analysis and visualization, and (7) data integration solutions. Detailed descriptions of functions and requirements are provided to guide customized data analysis workflows.

MARS: A Multipurpose Software for Untargeted LC-MS-Based Metabolomics and Exposomics.

Untargeted metabolomics is a growing field, in which recent advances in high-resolution mass spectrometry coupled with liquid chromatography (LC-MS) have facilitated untargeted approaches as a result of improvements in sensitivity, mass accuracy, and resolving power. However, a very large amount of data are generated. Consequently, using computational tools is now mandatory for the in-depth analysis of untargeted metabolomics data. This article describes MetAbolomics ReSearch (MARS), an all-in-one vendor-agnostic graphical user interface-based software applying LC-MS analysis to untargeted metabolomics. All of the analytical steps are described (from instrument data conversion and processing to statistical analysis, annotation/identification, quantification, and preliminary biological interpretation), and tools developed to improve annotation accuracy (e.g., multiple adducts and in-source fragmentation detection, trends across samples, and the MS/MS validator) are highlighted. In addition, MARS allows in-house building of reference databases, to bypass the limits of freely available MS/MS spectra collections. Focusing on the flexibility of the software and its user-friendliness, which are two important features in multipurpose software, MARS could provide new perspectives in untargeted metabolomics data analysis.

Development of an easy-to-set-up multiple heart-cutting achiral-chiral LC-LC method for the analysis of branched-chain amino acids in commercial tablets.

In this paper, the development and application of a multiple heart-cutting achiral-chiral LC-LC method (mLC-LC) for the analysis of dansylated (Dns) branched-chain amino acids in commercial tablets are described. In the first dimension, a Waters Xbridge RP C18 achiral column was used under gradient conditions with buffered aqueous solution and acetonitrile. The elution order Dns-valine (Dns-Val) < Dns-isoleucine (Dns-Ile) < Dns-leucine (Dns-Leu) turned out with full resolution between adjacent peaks: 7.25 and 1.50 for the Val/Ile and the Ile/Leu pairs, respectively. A "research" validation study was performed, revealing high accuracy (Recovery%) and precision (RSD%) using two external set solutions, respectively, in the range 93.7%-104.1% and 0.4%-3.2%. The C18 column was connected via a two-position six-port switching valve to the quinidine-based Chiralpak quinidine-anion-exchange chiral column. A water/acetonitrile, 30/70 (v/v) with 50 mM ammonium acetate (apparent pH of 5.5) eluent allowed getting the three enantiomers' pairs resolved: RS equal to 4.3 for Dns-Val and Dns-Ile, and 1.7 for Dns-Leu. The application of the mLC-LC method confirmed that the content of Val, Ile, and Leu in the tablets was compliant with that labeled by the producer. Only l-enantiomers were found in the food supplement, as confirmed by LC-MS/MS analysis.

Plasma lipidomics analysis reveals altered profile of triglycerides and phospholipids in children with Medium-Chain Acyl-CoA dehydrogenase deficiency.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most prevalent mitochondrial fatty acid ß-oxidation disorder. In this study, we assessed the variability of the lipid profile in MCADD by analysing plasma samples obtained from 25 children with metabolically controlled MCADD (following a normal diet with frequent feeding and under l-carnitine supplementation) and 21 paediatric control subjects (CT). Gas chromatography-mass spectrometry was employed for the analysis of esterified fatty acids, while high-resolution C18-liquid chromatography-mass spectrometry was used to analyse lipid species. We identified a total of 251 lipid species belonging to 15 distinct lipid classes. Principal component analysis revealed a clear distinction between the MCADD and CT groups. Univariate analysis demonstrated that 126 lipid species exhibited significant differences between the two groups. The lipid species that displayed the most pronounced variations included triacylglycerols and phosphatidylcholines containing saturated and monounsaturated fatty acids, specifically C14:0 and C16:0, which were found to be more abundant in MCADD. The observed changes in the plasma lipidome of children with non-decompensated MCADD suggest an underlying alteration in lipid metabolism. Therefore, longitudinal monitoring and further in-depth investigations are warranted to better understand whether such alterations are specific to MCADD children and their potential long-term impacts.

Use of ionic liquids in amidation reactions for proteolysis targeting chimera synthesis.

Selective degradation of disease-causing proteins using proteolysis targeting chimeras (PROTACs) has gained great attention, thanks to its several advantages over traditional therapeutic modalities. Despite the advances made so far, the structural chemical complexity of PROTACs poses challenges in their synthetic approaches. PROTACs are typically prepared through a convergent approach, first synthesizing two fragments separately (target protein and E3 ligase ligands) and then coupling them to produce a fully assembled PROTAC. The amidation reaction represents the most common coupling exploited in PROTACs synthesis. Unfortunately, the overall isolated yields of such synthetic procedures are usually low due to one or more purification steps to obtain the final PROTAC with acceptable purity. In this work, we focused our attention on the optimization of the final amidation step for the synthesis of an anti-SARS-CoV-2 PROTAC by investigating different amidation coupling reagents and a range of alternative solvents, including ionic liquids (ILs). Among the ILs screened, [OMIM][ClO4] emerged as a successful replacement for the commonly used DMF within the HATU-mediated amidation reaction, thus allowing the synthesis of the target PROTAC under mild and sustainable conditions in very high isolated yields. With the optimised conditions in hand, we explored the scalability of the synthetic approach and the substrate scope of the reaction by employing different E3 ligase ligand (VHL and CRBN)-based intermediates containing linkers of different lengths and compositions or by using different target protein ligands. Interestingly, in all cases, we obtained high isolated yields and complete conversion in short reaction times.

Whole Blood and Plasma-Based Lipid Profiling Reveals Distinctive Metabolic Changes in Systemic Lupus Erythematosus and Systemic Sclerosis.

Autoimmune diseases (AID), such as systemic lupus erythematosus (SLE) and systemic sclerosis (SS), are complex conditions involving immune system dysregulation. Diagnosis is challenging, requiring biomarkers for improved detection and prediction of relapses. Lipids have emerged as potential biomarkers due to their role in inflammation and immune response. This study uses an untargeted C18 RP-LC-MS lipidomics approach to comprehensively assess changes in lipid profiles in patients with SLE and SS. By analyzing whole blood and plasma, the study aims to simplify the lipidomic analysis, explore cellular-level lipids, and compare lipid signatures of SLE and SS with healthy controls. Our findings showed variations in the lipid profile of SLE and SS. Sphingomyelin and ceramide molecular species showed significant increases in plasma samples from SS patients, suggesting an atherosclerotic profile and potentially serving as lipid biomarkers. Phosphatidylserine species in whole blood from SLE patients exhibited elevated levels supporting previously reported dysregulated processes of cell death and defective clearance of dying cells in this AID. Moreover, decreased phospholipids bearing PUFA were observed, potentially attributed to the degradation of these species through lipid peroxidation processes. Further studies are needed to better understand the role of lipids in the pathological mechanisms underlying SLE and SS.

A platelet lipidomics signature in patients with COVID-19.

Ischemic cardiovascular and venous thromboembolic events are a frequent cause of death in severe COVID-19 patients. Platelet activation plays a key role in these complications, however platelet lipidomics have not been studied yet. The aim of our pilot investigation was to perform a preliminary study of platelet lipidomics in COVID-19 patients compared to healthy subjects. Lipid extraction and identification of ultrapurified platelets from eight hospitalized COVID-19 patients and eight age- and sex-matched healthy controls showed a lipidomic pattern almost completely separating COVID-19 patients from healthy controls. In particular, a significant decrease of ether phospholipids and increased levels of ganglioside GM3 were observed in platelets from COVID-19 patients. In conclusion, our study shows for the first time that platelets from COVID-19 patients display a different lipidomics signature distinguishing them from healthy controls, and suggests that altered platelet lipid metabolism may play a role in viral spreading and in the thrombotic complications of COVID-19.

What is the context? Besides respiratory system involvement, venous thromboembolism is a severe complication of COVID-19, largely due to the strong derangement of hemostasis, with platelets playing a central role.Great attention has recently been devoted to lipid alterations in COVID-19, both because viruses by reprogramming cellular lipid metabolism remodel lipid membranes to fuel their replication, and because the COVID-19-associated cytokine storm may affect cell/plasma lipidomic signatures.Lipidomics studies in COVID-19 patients have been performed mainly in plasma and serum.To the best of our knowledge, platelet lipidomics have not been examined despite the central role played by platelets in COVID-19 complications.What is the aim of the study?The aim of our pilot study was to preliminarily explore whether platelet lipidomics is altered in COVID-19 patients compared to age- and sex-matched healthy subjects, analyzing lipidomic profile of ultrapurified platelets.What are the results of our study? Our study shows for the first time that platelets from COVID-19 patients display a different lipidomics signature distinguishing them from healthy controls.Ether phospholipids and, intriguingly, two phytoceramides were lower, while ganglioside GM3 was higher in COVID-19 samples compared to healthy controls.What is the impact?Despite the small number of COVID-19 patients enrolled, recognized as a limitation of our study, we show, for the first time, that platelets from COVID-19 patients present a different lipidomics signature and suggest that altered platelet lipid metabolism may play a significant role in viral spreading and in the thrombotic complications of COVID-19.

CROMATIC: Cross-Relationship Map of Cavities from Coronaviruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of COVID-19 disease, has rapidly imposed an urgent need to identify effective drug candidates. In this context, the high resolution and non-redundant beta-Coronavirus protein cavities database is pivotal to help virtual screening protocols. Furthermore, the cross-relationship among cavities can lead to highlighting multitarget therapy chances. Here, we first collect all protein cavities on SARS-CoV-2, SARS-CoV, and MERS-CoV X-ray structures, and then, we compute a similarity map by using molecular interaction fields (MIFs). All the results come together in CROMATIC (CROss-relationship MAp of CaviTIes from Coronaviruses). CROMATIC encloses both a comprehensive and a non-redundant version of the cavities collection and a similarity map revealing, on the one hand, cavities that are conserved among the three Coronaviruses and, on the other hand, unexpected similarities among cavities that can represent a key starting point for multitarget therapy strategies. Similarity analysis was also performed for the available structures of SARS-CoV-2 spike variants, linking sequence mutations to three-dimensional interaction alterations. The CROMATIC repository is freely available to the scientific community at https://github.com/moldiscovery/sars-cromatic.

Synthesis and biological activity of cyclopropyl Δ7-dafachronic acids as DAF-12 receptor ligands.

The four cyclopropyl stereoisomers of Δ7-dafachronic acids were prepared from the bile acid hyodeoxycholic acid and employed as chemical tools to exploit the importance of the orientation and spatial disposition of the carboxyl tail and the C25-methyl group for the binding at the DAF-12 receptor. The synthesis route was based on (a) Walden inversion and stereoselective PtO2-hydrogenation to convert the L-shaped 5ß-cholanoid scaffold into the planar 5α-sterol intermediate; (b) two-carbon homologation of the side chain by Wittig and cyclopropanation reaction; and (c) formation of the 3-keto group and Δ7 double bond. The synthesized isomers were isolated and tested for their activity as DAF-12 ligands by AlphaScreen assays. Results showed a significant loss of potency and efficacy for all the four stereoisomers when compared to the parent endogenous ligand. Computational analysis has evidenced the configurational and conformational arrangement of both the carboxylic and the C25-methyl group of dafachronic acids as key structural determinants for DAF-12 binding and activation.

Automatic Identification of Lansoprazole Degradants under Stress Conditions by LC-HRMS with MassChemSite and WebChembase.

Stress testing is one of the most important parts of the drug development process, helping to foresee stability problems and to identify degradation products. One of the processes involving stress testing is represented by forced degradation studies, which can predict the impact of certain conditions of pH, moisture, heat, or other negative effects due to transportation or packaging issues on drug potency and purity, ensuring patient safety. Regulatory agencies have been working on a standardization of laboratory procedures since the past two decades. One of the results of those years of intensive research is the International Conference on Harmonization (ICH) guidelines, which clearly define which forced degradation studies should be performed on new drugs, which become a routine work in pharmaceutical laboratories. Since used techniques based on high-performance liquid chromatography coupled with high-resolution mass spectrometry have been developed years ago and are now mastered by pharmaceutical scientists, automation of data analysis, and thus data processing, is becoming a hot topic nowadays. In this work, we present MassChemSite and WebChembase as a tandem to automatize the routine analysis studies without missing information quality, using as a case study the degradation of lansoprazole under acidic, oxidative, basic, and neutral stress conditions.

A Novel Lipidomics-Based Approach to Evaluating the Risk of Clinical Hepatotoxicity Potential of Drugs in 3D Human Microtissues.

The importance of adsorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis is expected to grow substantially due to recent failures in detecting severe toxicity issues of new chemical entities during preclinical/clinical development. Traditionally, safety risk assessment studies for humans have been conducted in animals during advanced preclinical or clinical phase of drug development. However, potential drug toxicity in humans now needs to be detected in the drug discovery process as soon as possible without reliance on animal studies. The "omics", such as genomics, proteomics, and metabolomics, have recently entered pharmaceutical research in both drug discovery and drug development, but to the best of our knowledge, no applications in high-throughput safety risk assessment have been attempted so far. This paper reports an innovative method to anticipate adverse drug effects in an early discovery phase based on lipid fingerprints using human three-dimensional microtissues. The risk of clinical hepatotoxicity potential was evaluated for a data set of 22 drugs belonging to five different therapeutic chemical classes and with various drug-induced liver injury effect. The treatment of microtissues with repeated doses of each drug allowed collecting lipid fingerprints for five time points (2, 4, 7, 9, and 11 days), and multivariate statistical analysis was applied to search for correlations with the hepatotoxic effect. The method allowed clustering of the drugs based on their hepatotoxic effect, and the observed lipid impairments for a number of drugs was confirmed by literature sources. Compared to traditional screening methods, here multiple interconnected variables (lipids) are measured simultaneously, providing a snapshot of the cellular status from the lipid perspective at a molecular level. Applied here to hepatotoxicity, the proposed workflow can be applied to several tissues, being tridimensional microtissues from various origins.

Structure-metabolism relationships in human-AOX: Chemical insights from a large database of aza-aromatic and amide compounds.

Aldehyde oxidase (AOX) is a metabolic enzyme catalyzing the oxidation of aldehyde and aza-aromatic compounds and the hydrolysis of amides, moieties frequently shared by the majority of drugs. Despite its key role in human metabolism, to date only fragmentary information about the chemical features responsible for AOX susceptibility are reported and only "very local" structure-metabolism relationships based on a small number of similar compounds have been developed. This study reports a more comprehensive coverage of the chemical space of structures with a high risk of AOX phase I metabolism in humans. More than 270 compounds were studied to identify the site of metabolism and the metabolite(s). Both electronic [supported by density functional theory (DFT) calculations] and exposure effects were considered when rationalizing the structure-metabolism relationship.

Delving into the Polar Lipidome by Optimized Chromatographic Separation, High-Resolution Mass Spectrometry, and Comprehensive Identification with Lipostar: Microalgae as Case Study.

The work describes the chromatographic separation optimization of polar lipids on Kinetex-EVO, particularly focusing on sulfolipids in spirulina microalgae ( Arthrospira platensis). Gradient shape and mobile-phase modifiers (pH and buffer) were tested on lipid standards. Different conditions were evaluated, and resolution, peak capacity, and peak shape were calculated both in negative mode, for sulfolipids and phospholipids, and in positive mode, for glycolipids. A high-confidence lipid identification strategy was also applied. In collaboration with software creators and developers, Lipostar was implemented to improve the identification of phosphoglycerolipids and to allow the identification of glycosylmonoradyl- and glycosyldiradyl-glycerols classes, the last being the main focus of this work. By this approach, an untargeted screening also for searching lipids not yet reported in the literature could be accomplished. The optimized chromatographic conditions and database search were tested for lipid identification first on the standard mixture, then on the polar lipid extract of spirulina microalgae, for which 205 lipids were identified.

Lipostar, a Comprehensive Platform-Neutral Cheminformatics Tool for Lipidomics.

To date, the main limitations for LC-MS-based untargeted lipidomics reside in the lack of adequate computational and cheminformatics tools that are able to support the analysis of several thousands of species from biological samples, enabling data mining and automating lipid identification and external prediction processes. To address these issues, we developed Lipostar, novel vendor-neutral high-throughput software that effectively supports both targeted and untargeted LC-MS lipidomics, implementing data acquisition, user-friendly multivariate analysis (to be used for model generation and new sample predictions), and advanced lipid identification protocols that can work with or without the support of preformed lipid databases. Moreover, Lipostar integrates the lipidomic processes with a full metabolite identification (MetID) procedure, essential in drug safety applications and in translational studies. Case studies demonstrating a number of Lipostar features are also presented.

Efficient and regioselective one-step synthesis of 7-aryl-5-methyl- and 5-aryl-7-methyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidine derivatives.

Two facile and efficient one-step procedures for the regioselective synthesis of 7-aryl-5-methyl- and 5-aryl-7-methyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines have been developed, via reactions of 3,5-diamino-1,2,4-triazole with variously substituted 1-aryl-1,3-butanediones and 1-aryl-2-buten-1-ones, respectively. The excellent yield and/or regioselectivity shown by the reactions decreased when ethyl 5-amino-1,2,4-triazole-3-carboxylate was used. [1,2,4]Triazolo[1,5-a]pyrimidine being a privileged scaffold, the procedures herein reported may be useful for the preparation of biologically active compounds. In this study, the preparation of a set of compounds based on the [1,2,4]triazolo[1,5-a]pyrimidine scaffold led to the identification of compound 20 endowed with a very promising ability to inhibit influenza virus RNA polymerase PA-PB1 subunit heterodimerization.

Passive Intestinal Absorption of Representative Plant Secondary Metabolites: A Physicochemical Study.

Natural products are generally ingested as part of traditional herbal decoctions or in the current diet. However, in natural product research, the bioavailability of secondary metabolites is often poorly investigated. In this work, a systematic study was carried out in order to highlight the physicochemical parameters that mainly influence the passive intestinal absorption of natural products. For this, a representative set of natural products including alkaloids, coumarins, flavonoid aglycones and glycosides, and carboxylic acids was selected and their physicochemical properties were predicted using relevant Volsurf+ descriptors. The chemical space obtained with this unbiased method was then correlated with experimental passive intestinal permeability data, which highlighted the main influence of lipophilicity, global hydrophilicity, size, and the ionisation state on passive intestinal absorption of natural products. Since the pH range encountered in the intestine is wide, the influence of the ionisation was investigated deeper experimentally. The ionisation state of weakly ionisable natural products, such as flavonoid aglycones, alkaloids, and carboxylic acids, was found to prevent the passive intestinal absorption of such natural products completely. In addition, the impact of solubility issues on passive permeability results was evaluated in cases of poorly water-soluble natural products, such as flavonoid aglycones and coumarins. The biomimetic fasted state simulated fluid-version 2 was found to improve the apparent solubility of such poorly soluble natural products without influencing their permeability behaviours. The use of such a solubilising buffer was found to be well adapted to the hexadecane membrane-parallel artificial membrane permeability assay and can circumvent the solubility issues encountered with poorly soluble natural products in such an assay.

Use of lipidomics to investigate sebum dysfunction in juvenile acne.

Acne is a multifactorial skin disorder frequently observed during adolescence with different grades of severity. Multiple factors centering on sebum secretion are implicated in acne pathogenesis. Despite the recognized role of sebum, its compositional complexity and limited analytical approaches have hampered investigation of alterations specifically associated with acne. To examine the profiles of lipid distribution in acne sebum, 61 adolescents (29 males and 32 females) were enrolled in this study. Seventeen subjects presented no apparent clinical signs of acne. The 44 affected individuals were clinically classified as mild (13 individuals), moderate (19 individuals), and severe (12 individuals) acne. Sebum was sampled from the forehead with Sebutape(TM) adhesive patches. Profiles of neutral lipids were acquired with rapid-resolution reversed-phase/HPLC-TOF/MS in positive ion mode. Univariate and multivariate statistical analyses led to the identification of lipid species with significantly different levels between healthy and acne sebum. The majority of differentiating lipid species were diacylglycerols (DGs), followed by fatty acyls, sterols, and prenols. Overall, the data indicated an association between the clinical grading of acne and sebaceous lipid fingerprints and highlighted DGs as more abundant in sebum from adolescents affected with acne.