Profiling Intact Glycosphingolipids with Automated Structural Annotation and Quantitation from Human Samples with Nanoflow Liquid Chromatography Mass Spectrometry.

Sphingolipids are an essential subset of bioactive lipids found in most eukaryotic cells that contribute to membrane biophysical properties and are involved in cellular differentiation, recognition, and mediating interactions. The described nanoHPLC-ESI-Q/ToF methodology utilizes known biosynthetic pathways, accurate mass detection, optimized collision-induced disassociation, and a robust nanoflow chromatographic separation for the analysis of intact sphingolipids found in human tissue, cells, and serum. The methodology was developed and validated with an emphasis on addressing the common issues experienced in profiling these amphipathic lipids, which are part of the glycocalyx and lipidome. The high sensitivity obtained using nanorange flow rates with robust chromatographic reproducibility over a wide range of concentrations and injection volumes results in confident identifications for profiling these low-abundant biomolecules.

Integrating Computational Methods in Network Pharmacology and In Silico Screening to Uncover Multi-targeting Phytochemicals against Aberrant Protein Glycosylation in Lung Cancer.

Glycoproteins are an underexploited drug target for cancer therapeutics. In this work, we integrated computational methods in network pharmacology and in silico docking approaches to identify phytochemical compounds that could potentially interact with several cancer-associated glycoproteins. We first created a database of phytochemicals from selected plant species, Manilkara zapota (sapodilla/chico), Mangifera indica (mango), Annona muricata (soursop/guyabano), Artocarpus heterophyllus (jackfruit/langka), Lansium domesticum (langsat/lanzones), and Antidesma bunius (bignay), and performed pharmacokinetic analysis to determine their drug-likeness properties. We then constructed a phytochemical-glycoprotein interaction network and characterized the degree of interactions between the phytochemical compounds and with cancer-associated glycoproteins and other glycosylation-related proteins. We found a high degree of interactions from α-pinene (Mangifera indica), cyanomaclurin (Artocarpus heterophyllus), genistein (Annona muricata), kaempferol (Annona muricata and Antidesma bunius), norartocarpetin (Artocarpus heterophyllus), quercetin (Annona muricata, Antidesma bunius, Manilkara zapota, Mangifera indica), rutin (Annona muricata, Antidesma bunius, Lansium domesticum), and ellagic acid (Antidesma bunius and Mangifera indica). Subsequent docking analysis confirmed that these compounds could potentially bind to EGFR, AKT1, KDR, MMP2, MMP9, ERBB2, IGF1R, MTOR, and HRAS proteins, which are known cancer biomarkers. In vitro cytotoxicity assays of the plant extracts showed that the n-hexane, ethyl acetate, and methanol leaf extracts from A. muricata, L. domesticum and M. indica gave the highest growth inhibitory activity against A549 lung cancer cells. These may help further explain the reported cytotoxic activities of select compounds from these plant species.

Comparative proteomics reveals anticancer compounds from Lansium domesticum against NSCLC cells target mitochondrial processes.

Lansium domesticum is identified as a potential source of anticancer compounds. However, there are minimal studies on its anti-lung cancer properties as well as its mechanism of action. Here, we show the specificity of lanzones hexane (LH) leaf extracts to non-small cell lung cancer cells (A549) compared to normal lung fibroblast cells (CCD19-Lu) and normal epithelial prostate cells (PNT2). Subsequent bioassay-guided fractionation of the hexane leaf extracts identified two bioactive fractions with IC50 values of 2.694 µg/ml (LH6-6) and 2.883 µg/ml (LH7-6). LH 6-6 treatment (1 µg/ml concentration) also showed a significantly reduced migration potential of A549 relative to the control. Thirty-one phytocompounds were isolated and identified using gas chromatography-mass spectrometric (MS) analysis and were then subjected to network pharmacology analysis to assess its effects on lung cancer target proteins. Using liquid chromatography-tandem mass spectrometry proteomics experiments, we were able to show that these compounds cause cytotoxic effects through targeting mitochondrial processes in A549 lung cancer cells.

In silico screening-based discovery of inhibitors against glycosylation proteins dysregulated in cancer.

Targeting enzymes associated with the biosynthesis of aberrant glycans is an under-utilized strategy in discovering potential inhibitors or drugs against cancer. The formation of cancer-associated glycans is mainly due to the dysregulated expression of glycosyltransferases and glycosidases, which play crucial roles in maintaining cellular structure and function. We screened a database of more than 14,000 compounds consisting of natural products and drugs for inhibition against four glycosylation enzymes - Alpha1-6FucT, ST6Gal1, ERMan1, and GlcNAcT-V. The top inhibitors identified against each enzyme were subsequently analyzed for potential binding against all four enzymes. In silico screening results show several promising candidates that could potentially inhibit all four enzymes: (1) Amb20622156 (demethylwedelolactone) [ERMan1: -9.3 kcal/mol; Alpha1-6FucT: -7.3 kcal/mol; ST6Gal1: -8.4 kcal/mol; GlcNAcT-V: -7.2 kcal/mol], (2) Amb22173588 (1,2-dihydrotanshinone I) [ERMan1: -9.3 kcal/mol; Alpha1-6FucT: -6.1 kcal/mol; ST6Gal1: -9.2 kcal/mol; GlcNAcT-V: -7.9 kcal/mol], and (3) Amb22173591 (tanshinol B) [ERMan1: -9.3 kcal/mol; Alpha1-6FucT: -6.0 kcal/mol; ST6Gal1: -9.8 kcal/mol; GlcNAcT-V: -7.7 kcal/mol]. Drug-enzyme active site residue interaction analyses show that the putative inhibitors form non-covalent bonding interactions with key active site residues in each enzyme, suggesting critical target residues in the four enzymes' active sites. Furthermore, pharmacokinetic property prediction analysis using pkCSM indicates that all of these inhibitors have good ADMETox properties (i.e., log P < 5, Caco-2 permeability > 0.90, intestinal absorption > 30%, skin permeability>-2.5, CNS permeability <-3, maximum tolerated dose < 0.477, minnow toxicity<-0.3). The in silico docking approach to glycosylation enzyme inhibitor prediction could help guide and streamline the discovery of novel inhibitors against enzymes involved in aberrant protein glycosylation.Communicated by Ramaswamy H. Sarma.

Biological Assay-Guided Fractionation and Mass Spectrometry-Based Metabolite Profiling of Annona muricata L. Cytotoxic Compounds against Lung Cancer A549 Cell Line.

Annona muricata L. (Guyabano) leaves are reported to exhibit anticancer activity against cancer cells. In this study, the ethyl acetate extract from guyabano leaves was purified through column chromatography, and the cytotoxic effects of the semi-purified fractions were evaluated against A549 lung cancer cells using in vitro MTS cytotoxicity and scratch/wound healing assays. Fractions F15-16C and F15-16D exhibited the highest anticancer activity in the MTS assay, with % cytotoxicity values of 99.6% and 99.4%, respectively. The bioactivity of the fractions was also consistent with the results of the scratch/wound healing assay. Moreover, untargeted metabolomics was employed on the semi-purified fractions to determine the putative compounds responsible for the bioactivity. The active fractions were processed using LC-MS/MS analysis with the integration of the following metabolomic tools: MS-DIAL (for data processing), MetaboAnalyst (for data analysis), GNPS (for metabolite annotation), and Cytoscape (for network visualization). Results revealed that the putative compounds with a significant difference between active and inactive fractions in PCA and OPLS-DA models were pheophorbide A and diphenylcyclopropenone.

An Integrated Mass Spectrometry-Based Glycomics-Driven Glycoproteomics Analytical Platform to Functionally Characterize Glycosylation Inhibitors.

Cancer progression is linked to aberrant protein glycosylation due to the overexpression of several glycosylation enzymes. These enzymes are underexploited as potential anticancer drug targets and the development of rapid-screening methods and identification of glycosylation inhibitors are highly sought. An integrated bioinformatics and mass spectrometry-based glycomics-driven glycoproteomics analysis pipeline was performed to identify an N-glycan inhibitor against lung cancer cells. Combined network pharmacology and in silico screening approaches were used to identify a potential inhibitor, pictilisib, against several glycosylation-related proteins, such as Alpha1-6FucT, GlcNAcT-V, and Alpha2,6-ST-I. A glycomics assay of lung cancer cells treated with pictilisib showed a significant reduction in the fucosylation and sialylation of N-glycans, with an increase in high mannose-type glycans. Proteomics analysis and in vitro assays also showed significant upregulation of the proteins involved in apoptosis and cell adhesion, and the downregulation of proteins involved in cell cycle regulation, mRNA processing, and protein translation. Site-specific glycoproteomics analysis further showed that glycoproteins with reduced fucosylation and sialylation were involved in apoptosis, cell adhesion, DNA damage repair, and chemical response processes. To determine how the alterations in N-glycosylation impact glycoprotein dynamics, modeling of changes in glycan interactions of the ITGA5-ITGB1 (Integrin alpha 5-Integrin beta-1) complex revealed specific glycosites at the interface of these proteins that, when highly fucosylated and sialylated, such as in untreated A549 cells, form greater hydrogen bonding interactions compared to the high mannose-types in pictilisib-treated A549 cells. This study highlights the use of mass spectrometry to identify a potential glycosylation inhibitor and assessment of its impact on cell surface glycoprotein abundance and protein-protein interaction.