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.

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.

Design, synthesis, and biological evaluation of first-in-class indomethacin-based PROTACs degrading SARS-CoV-2 main protease and with broad-spectrum antiviral activity.

To date, Proteolysis Targeting Chimera (PROTAC) technology has been successfully applied to mediate proteasomal-induced degradation of several pharmaceutical targets mainly related to oncology, immune disorders, and neurodegenerative diseases. On the other hand, its exploitation in the field of antiviral drug discovery is still in its infancy. Recently, we described two indomethacin (INM)-based PROTACs displaying broad-spectrum antiviral activity against coronaviruses. Here, we report the design, synthesis, and characterization of a novel series of INM-based PROTACs that recruit either Von-Hippel Lindau (VHL) or cereblon (CRBN) E3 ligases. The panel of INM-based PROTACs was also enlarged by varying the linker moiety. The antiviral activity resulted very susceptible to this modification, particularly for PROTACs hijacking VHL as E3 ligase, with one piperazine-based compound (PROTAC 6) showing potent anti-SARS-CoV-2 activity in infected human lung cells. Interestingly, degradation assays in both uninfected and virus-infected cells with the most promising PROTACs emerged so far (PROTACs 5 and 6) demonstrated that INM-PROTACs do not degrade human PGES-2 protein, as initially hypothesized, but induce the concentration-dependent degradation of SARS-CoV-2 main protease (Mpro) both in Mpro-transfected and in SARS-CoV-2-infected cells. Importantly, thanks to the target degradation, INM-PROTACs exhibited a considerable enhancement in antiviral activity with respect to indomethacin, with EC50 values in the low-micromolar/nanomolar range. Finally, kinetic solubility as well as metabolic and chemical stability were measured for PROTACs 5 and 6. Altogether, the identification of INM-based PROTACs as the first class of SARS-CoV-2 Mpro degraders demonstrating activity also in SARS-CoV-2-infected cells represents a significant advance in the development of effective, broad-spectrum anti-coronavirus strategies.

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.

Discovery and Structure-Activity Relationships of Novel ssDAF-12 Receptor Modulators.

The nuclear receptor ssDAF-12 has been recognized as the key molecular player regulating the life cycle of the nematode parasite Strongyloides stercoralis. ssDAF-12 ligands permit the receptor to function as an on/off switch modulating infection, making it vulnerable to therapeutic intervention. In this study, we report the design and synthesis of a set of novel dafachronic acid derivatives, which were used to outline the first structure-activity relationship targeting the ssDAF-12 receptor and to unveil hidden properties shared by the molecular shape of steroidal ligands that are relevant to the receptor binding and modulation. Moreover, biological results led to the discovery of sulfonamide 3 as a submicromolar ssDAF-12 agonist endowed with a high receptor selectivity, no toxicity, and improved properties, as well as to the identification of unprecedented ssDAF-12 antagonists that can be exploited in the search for novel chemical tools and alternative therapeutic approaches for treating parasitism such as Strongyloidiasis.

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.

VHL-Modified PROteolysis TArgeting Chimeras (PROTACs) as a Strategy to Evade Metabolic Degradation in In Vitro Applications.

PROteolysis TArgeting Chimeras (PROTACs) are tripartite molecules consisting of a linker connecting a ligand for a protein of interest to an E3 ligase recruiter, whose rationale relies on proteasome-based protein degradation. PROTACs have expanded as a therapeutic strategy to open new avenues for unmet medical needs. Leveraging our expertise, we undertook a series of in vitro experiments aimed at elucidating PROTAC metabolism. In particular, we focused on PROTACs recruiting the von Hippel-Lindau (VHL) E3 ligase. After high-resolution mass spectrometry measurements, a characteristic metabolite with mass reduction of 200 units was detected and successively confirmed as a product deriving from the cleavage of the VHL ligand moiety. Subsequently, we identified hepatic and extrahepatic prolyl endopeptidases as the main putative metabolic enzymes involved. Finally, we designed and synthesized analogs of the VHL ligands that we further exploited for the synthesis of novel VHL-directed PROTACs with an improved metabolic stability in in vitro applications.

Investigating Performance of the SLIM-Based High Resolution Ion Mobility Platform for Separation of Isomeric Phosphatidylcholine Species.

Lipids are structurally diverse molecules that play a pivotal role in a plethora of biological processes. However, deciphering the biological roles of the specific lipids is challenging due to the existence of numerous isomers. This high chemical complexity of the lipidome is one of the major challenges in lipidomics research, as the traditional liquid chromatography-mass spectrometry (LC-MS) based approaches are often not powerful enough to resolve these isomeric and isobaric nuances within complex samples. Thus, lipids are uniquely suited to the benefits provided by multidimensional liquid chromatography-ion mobility-mass spectrometry (LC-IM-MS) analysis. However, many forms of lipid isomerism, including double-bond positional isomers and regioisomers, are structurally similar such that their collision cross section (CCS) differences are unresolvable via conventional IM approaches. Here we evaluate the performance of a high resolution ion mobility (HRIM) system based on structures for lossless ion manipulation (SLIM) technology interfaced to a high resolution quadrupole time-of-flight (QTOF) analyzer to address the noted lipidomic isomerism challenge. SLIM implements the traveling wave ion mobility technique along an ∼13 m ion path, providing longer path lengths to enable improved separation of isomeric features. We demonstrate the power of HRIM-MS to dissect isomeric PC standards differing only in double bond (DB) and stereospecific number (SN) positions. The partial separation of protonated DB isomers is significantly enhanced when they are analyzed as metal adducts. For sodium adducts, we achieve close to baseline separation of three different PC 18:1/18:1 isomers with different cis-double bond locations. Similarly, PC 18:1/18:1 (cis-9) can be resolved from the corresponding PC 18:1/18:1 (trans-9) form. The separation capacity is further enhanced when using silver ion doping, enabling the baseline separation of regioisomers that cannot be resolved when measured as sodium adducts. The sensitivity and reproducibility of the approach were assessed, and the performance for more complex mixtures was benchmarked by identifying PC isomers in total brain and liver lipid extracts.

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.

Analysis of Phosphatidylinositol Modifications by Reactive Nitrogen Species Using LC-MS: Coming to Grips with Their Nitroxidative Susceptibility.

Phosphatidylinositols (PIs) are complex lipids that play a key role in cell signaling. Like other phospholipids, they are esterified with unsaturated fatty acyl residues (FAs), making them susceptible to modification by reactive oxygen and nitrogen species (RNS). Recent studies using mass spectrometry (MS)-based lipidomics approaches have revealed that lipid nitration results in a plethora of structurally and chemically modified lipids (epilipids), including nitrated and nitroxidized derivatives of phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, and cardiolipins. However, there is a notable lack of knowledge regarding the characterization of RNS-modified PI derivatives. In this study, we used C18 high-resolution liquid chromatography-tandem MS approaches to describe the fragmentation signature of nitrated and nitroxidized PIs, bearing different fatty acyl chains. Using this approach and accurate mass measurements, we were able to identify nitro- PI derivatives, dinitro- and nitrohydroxy- derivatives for a few PI species. The data showed the typical neutral loss of nitrous acid (HNO2) as well as the fragmentation patterns corresponding to modified fatty acyl chains (such as NOx-RCOO-, [M - NOx-RCOOH - H]- and [M - NOx-RCOOH - C6H10O5 - H]-), making it possible to identify these epilipids. The susceptibility of PIs to nitration was also investigated, revealing that it depends exclusively on the chains of unsaturated FAs esterified in PI, showing a higher conversion rate for those with C18:1. Overall, the knowledge gathered in this study will contribute to the precise characterization of these epilipids in complex biological samples, offering new opportunities to unveil the pathophysiological roles of nitrated and nitroxidized PI derivatives at the cellular and tissue levels.

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.

MassChemSite for In-Depth Forced Degradation Analysis of PARP Inhibitors Olaparib, Rucaparib, and Niraparib.

Drugs must satisfy several protocols and tests before being approved for the market. Among them, forced degradation studies aim to evaluate drug stability under stressful conditions in order to predict the formation of harmful degradation products (DPs). Recent advances in LC-MS instrumentation have facilitated the structure elucidation of degradants, although a comprehensive data analysis still represents a bottle-neck due to the massive amount of data that can be easily generated. MassChemSite has been recently described as a promising informatics solution for LC-MS/MS and UV data analysis of forced degradation experiments and for the automated structural identification of DPs. Here, we applied MassChemSite to investigate the forced degradation of three poly(ADP-ribose) polymerase inhibitors (olaparib, rucaparib, and niraparib) under basic, acidic, neutral, and oxidative stress conditions. Samples were analyzed by UHPLC with online DAD coupled to high-resolution mass spectrometry. The kinetic evolution of the reactions and the influence of solvent on the degradation process were also assessed. Our investigation confirmed the formation of three DPs of olaparib and the wide degradation of the drug under the basic condition. Intriguingly, base-catalyzed hydrolysis of olaparib was greater when the content of aprotic-dipolar solvent in the mixture decreased. For the other two compounds, whose stability has been much less studied previously, six new degradants of rucaparib were identified under oxidative degradation, while niraparib emerged as stable under all stress conditions tested.

Inhibitors of ABCG2-mediated multidrug resistance: Lead generation through computer-aided drug design.

Human breast cancer resistance protein (BCRP), known also as ABCG2, plays a major role in multiple drug resistance (MDR) in tumor cells. Through this ABC transporter, cancer cells acquire the ability of resistance to structurally and functionally unrelated anticancer drugs. Nowadays, the design of ABCG2 inhibitors as potential agents to enhance the chemotherapy efficacy is an interesting strategy. In this context, we have used computer-aided drug design (CADD) based on available data of a large series of potent inhibitors from our groups as an approach in guiding the design of effective ABCG2 inhibitors. We report therein the results on the use of the FLAPpharm method to elucidate the pharmacophoric features of one of the ABCG2 binding sites involved in the regulation of the basal ATPase activity of the transporter. The predictivity of the model was evaluated by testing three predicted compounds which were found to induce high inhibitory activity of BCRP, in the nanomolar range for the best of them.

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.

New bioisosteric sulphur-containing choline kinase inhibitors with a tracked mode of action.

Since the identification of human choline kinase as a protein target against cancer progression, many compounds have been designed to inhibit its function and reduce the biosynthesis of phosphatidylcholine. Herein, we propose a series of bioisosteric inhibitors that are based on the introduction of sulphur and feature improved activity and lipophilic/hydrophilic balance. The evaluation of the inhibitory and of the antiproliferative properties of the PL (dithioethane) and FP (disulphide) libraries led to the identification of PL 48, PL 55 and PL 69 as the most active compounds of the series. Docking analysis using FLAP suggests that for hits to leads, binding mostly involves an interaction with the Mg2+ cofactor, or its destabilization. The most active compounds of the two series are capable of inducing apoptosis following the mitochondrial pathway and to significantly reduce the expression of anti-apoptotic proteins such as the Mcl-1. The fluorescence properties of the compounds of the PL library allowed the tracking of their mode of action, while PAINS (Pan Assays Interference Structures) filtration databases suggest the lack of any unspecific biological response.

Analytical and computational workflow for in-depth analysis of oxidized complex lipids in blood plasma.

Lipids are a structurally diverse class of biomolecules which can undergo a variety of chemical modifications. Among them, lipid (per)oxidation attracts most of the attention due to its significance in the regulation of inflammation, cell proliferation and death programs. Despite their apparent regulatory significance, the molecular repertoire of oxidized lipids remains largely elusive as accurate annotation of lipid modifications is complicated by their low abundance and often unknown, biological context-dependent structural diversity. Here, we provide a workflow based on the combination of bioinformatics and LC-MS/MS technologies to support identification and relative quantification of oxidized complex lipids in a modification type- and position-specific manner. The developed methodology is used to identify epilipidomics signatures of lean and obese individuals with and without type 2 diabetes. The characteristic signature of lipid modifications in lean individuals, dominated by the presence of modified octadecanoid acyl chains in phospho- and neutral lipids, is drastically shifted towards lipid peroxidation-driven accumulation of oxidized eicosanoids, suggesting significant alteration of endocrine signalling by oxidized lipids in metabolic disorders.

Retinoic acid-induced 1 gene haploinsufficiency alters lipid metabolism and causes autophagy defects in Smith-Magenis syndrome.

Smith-Magenis syndrome (SMS) is a neurodevelopmental disorder characterized by cognitive and behavioral symptoms, obesity, and sleep disturbance, and no therapy has been developed to alleviate its symptoms or delay disease onset. SMS occurs due to haploinsufficiency of the retinoic acid-induced-1 (RAI1) gene caused by either chromosomal deletion (SMS-del) or RAI1 missense/nonsense mutation. The molecular mechanisms underlying SMS are unknown. Here, we generated and characterized primary cells derived from four SMS patients (two with SMS-del and two carrying RAI1 point mutations) and four control subjects to investigate the pathogenetic processes underlying SMS. By combining transcriptomic and lipidomic analyses, we found altered expression of lipid and lysosomal genes, deregulation of lipid metabolism, accumulation of lipid droplets, and blocked autophagic flux. We also found that SMS cells exhibited increased cell death associated with the mitochondrial pathology and the production of reactive oxygen species. Treatment with N-acetylcysteine reduced cell death and lipid accumulation, which suggests a causative link between metabolic dyshomeostasis and cell viability. Our results highlight the pathological processes in human SMS cells involving lipid metabolism, autophagy defects and mitochondrial dysfunction and suggest new potential therapeutic targets for patient treatment.