GPR17 is a class A orphan GPCR that regulates myelination in the central nervous system. Inhibiting GPR17 is a potential therapeutic mechanism to induce remyelination. The limited understanding of the structural basis of GPR17 agonism and antagonism is a challenge for GPR17 drug discovery. We present novel cryo-EM structures of GPR17 with bound orthosteric antagonist or agonist/G protein heterotrimer with biophysical and cellular characterization to inform rational drug discovery. Both ligands bind towards a lateral edge of the orthosteric pocket, while an exceptionally long extracellular loop 2 occupies and limits access to this site. In contrast, pharmacological and photoaffinity studies indicate that endogenous oxysterol agonists of GPR17 bind allosterically. Characterization of oxysterol agonism versus a known orthosteric ligand in oligodendrocytes further supports an allosteric mechanism. Therefore, the design of more potent orthosteric ligands is limited by pocket size and accessibility. Targeting allosteric binding offers an alternative approach to GPR17 antagonism.

This study aimed to gain insights into the dynamic proteome changes and underlying molecular mechanisms of de/remyelination in a cuprizone model, a widely used preclinical model of multiple sclerosis (MS). Longitudinal sampling of control or cuprizone-treated mouse brains was executed at 6 time points over 6 weeks. Data analysis included 8489 quantified proteins. Differential proteomic and GO analyses revealed that 5.9% of the quantified proteome was altered, including reported and novel de/remyelination-relevant protein changes and underlying pathways. We found that oligodendrocyte proteins (Fa2h and Ugt8) were significantly changed during demyelination, suggesting that dysregulated sphingolipid metabolism in MS may stem from oligodendrocyte pathology. Importantly, we showed that the cholesterol biosynthesis pathway was the most enriched biological process in a subset of significantly changed proteins, where myelination was highly enriched. We further validated the changes in the cholesterol biosynthesis pathway through targeted GC-MS analysis of intermediate sterols, supporting the critical role of cholesterol biosynthesis in de/remyelination. Unexpectedly, changes of myelin-associated proteins, Mbp and Plp1, were minimal, while Ermn showed significant reduction tracking with demyelination, indicating that some myelin protein changes are more sensitive to demyelination. Together with a list of significantly altered proteins, the results of this study could benefit future remyelination research.

Phospholipase D (PLD) is a phospholipase enzyme responsible for hydrolyzing phosphatidylcholine into the lipid signaling molecule, phosphatidic acid, and choline. From a therapeutic perspective, PLD has been implicated in human cancer progression as well as a target for neurodegenerative diseases, including Alzheimer's. Moreover, knockdown of PLD rescues the ALS phenotype in multiple Drosophila models of ALS (amyotrophic lateral sclerosis) and displays modest motor benefits in an SOD1 ALS mouse model. To further validate whether inhibiting PLD is beneficial for the treatment of ALS, a brain penetrant small molecule inhibitor with suitable PK properties to test in an ALS animal model is needed. Using a combination of ligand-based drug discovery and structure-based design, a dual PLD1/PLD2 inhibitor was discovered that is single digit nanomolar in the Calu-1 cell assay and has suitable PK properties for in vivo studies. To capture the in vivo measurement of PLD inhibition, a transphosphatidylation pharmacodynamic LC-MS assay was developed, in which a dual PLD1/PLD2 inhibitor was found to reduce PLD activity by 15-20-fold.

ObjectiveParkinson disease (PD) has useful symptomatic treatments that do not slow the neurodegenerative process, and no significant disease‐modifying treatments are approved. A key therapeutic target in PD is α‐synuclein (αS), which is both genetically implicated and accumulates in Lewy bodies rich in vesicles and other lipid membranes. Reestablishing αS homeostasis is a central goal in PD. Based on previous lipidomic analyses, we conducted a mouse trial of a stearoyl–coenzyme A desaturase (SCD) inhibitor (“5b”) that prevented αS‐positive vesicular inclusions and cytotoxicity in cultured human neurons.MethodsOral dosing and brain activity of 5b were established in nontransgenic mice. 5b in drinking water was given to mice expressing wild‐type human αS (WT) or an amplified familial PD αS mutation (E35K + E46K + E61K ["3K"]) beginning near the onset of nigral and cortical neurodegeneration and the robust PD‐like motor syndrome in 3K. Motor phenotypes, brain cytopathology, and SCD‐related lipid changes were quantified in 5b‐ versus placebo‐treated mice. Outcomes were compared to effects of crossing 3K to SCD1−/− mice.Results5b treatment reduced αS hyperphosphorylation in E46K‐expressing human neurons, in 3K neural cultures, and in both WT and 3K αS mice. 5b prevented subtle gait deficits in WT αS mice and the PD‐like resting tremor and progressive motor decline of 3K αS mice. 5b also increased αS tetramers and reduced proteinase K‐resistant lipid‐rich aggregates. Similar benefits accrued from genetically deleting 1 SCD allele, providing target validation.InterpretationProlonged reduction of brain SCD activity prevented PD‐like neuropathology in multiple PD models. Thus, an orally available SCD inhibitor potently ameliorates PD phenotypes, positioning this approach to treat human α‐synucleinopathies. ANN NEUROL 2021;89:74–90

Myelin is composed primarily of lipids and diseases affecting myelin are associated with alterations in its lipid composition. However, correlation of the spatial (in situ) distribution of lipids with the disease-associated compositional and morphological changes is not well defined. Herein we applied high resolution matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS), immunohistochemistry (IHC), and liquid chromatography–electrospray ionization–mass spectrometry (LC-ESI-MS) to evaluate brain lipid alterations in the dysmyelinating shiverer (Shi) mouse and cuprizone (Cz) mouse model of reversible demyelination. MALDI-IMS revealed a decrease in the spatial distribution of sulfatide (SHexCer) species, SHexCer (d42:2), and a phosphatidylcholine (PC) species, PC (36:1), in white matter regions like corpus callosum (CC) both in the Shi mouse and Cz mouse model. Changes in these lipid species were restored albeit not entirely upon spontaneous remyelination after demyelination in the Cz mouse model. Lipid distribution changes correlated with the local morphological changes as confirmed by IHC. LC-ESI-MS analyses of CC extracts confirmed the MALDI-IMS derived reductions in SHexCer and PC species. These findings highlight the role of SHexCer and PC in preserving the normal myelin architecture and our experimental approaches provide a morphological basis to define lipid abnormalities relevant to myelin diseases.

Fibroblasts/myofibroblasts are the key effector cells responsible for excessive extracellular matrix (ECM) deposition and fibrosis progression in both idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) patient lungs, thus it is critical to understand the transcriptomic and proteomic programs underlying their fibrogenic activity. We conducted the first integrative analysis of the fibrotic programming in these cells at the levels of gene and microRNA (miRNA) expression, as well as deposited ECM protein to gain insights into how fibrotic transcriptional programs culminate in aberrant ECM protein production/deposition. We identified messenger RNA (mRNA), miRNA, and deposited matrisome protein signatures for IPF and SSc fibroblasts obtained from lung transplants using next-generation sequencing and mass spectrometry. SSc and IPF fibroblast transcriptional signatures were remarkably similar, with enrichment of WNT, TGF-β, and ECM genes. miRNA-seq identified differentially regulated miRNAs, including downregulation of miR-29b-3p, miR-138-5p and miR-146b-5p in disease fibroblasts and transfection of their mimics decreased expression of distinct sets of fibrotic signature genes as assessed using a Nanostring fibrosis panel. Finally, proteomic analyses uncovered a distinct "fibrotic" matrisome profile deposited by IPF and SSc fibroblasts compared to controls that highlights the dysregulated ECM production underlying their fibrogenic activities. Our comprehensive analyses of mRNA, miRNA, and matrisome proteomic profiles in IPF and SSc lung fibroblasts revealed robust fibrotic signatures at both the gene and protein expression levels and identified novel fibrogenesis-associated miRNAs whose aberrant downregulation in disease fibroblasts likely contributes to their fibrotic and ECM gene expression.

Delayed-release dimethyl fumarate (also known as gastro-resistant dimethyl fumarate), an oral therapeutic containing dimethyl fumarate (DMF) as the active ingredient, is currently approved for the treatment of relapsing multiple sclerosis. DMF is also a component in a distinct mixture product with 3 different salts of monoethyl fumarate (MEF), which is marketed for the treatment of psoriasis. Previous studies have provided insight into the pharmacologic properties of DMF, including modulation of kelch-like ECH-associated protein 1 (KEAP1), activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway, and glutathione (GSH) modulation; however, those of MEF remain largely unexplored. Therefore, the aim of this study was to evaluate the in vitro effects of DMF and MEF on KEAP1 modification, activation of the NRF2 pathway, and GSH conjugation. Using mass spectrometry, DMF treatment resulted in a robust modification of specific cysteine residues on KEAP1. In comparison, the overall degree of KEAP1 modification following MEF treatment was significantly less or undetectable. Consistent with KEAP1 cysteine modification, DMF treatment resulted in nuclear translocation of NRF2 and a robust transcriptional response in treated cells, as did MEF; however, the responses to MEF were of a lower magnitude or distinct compared to DMF. DMF was also shown to produce an acute concentration-dependent depletion of GSH; however, GSH levels eventually recovered and rose above baseline by 24 hours. In contrast, MEF did not cause acute reductions in GSH, but did produce an increase by 24 hours. Overall, these studies demonstrate that DMF and MEF are both pharmacologically active, but have differing degrees of activity as well as unique actions. These differences would be expected to result in divergent effects on downstream biology.

OBJECTIVE: To evaluate the effects of dimethyl fumarate (DMF) and monoethyl fumarate (MEF) on glutathione conjugation, Keap1 modification and activation of the Nrf2 pathway. BACKGROUND: Delayed-release DMF is approved in the U.S., Canada, and Australia for the treatment of multiple sclerosis. DMF is also one component of combination product with 3 different salts of MEF, and this combination is approved in Germany for the treatment of psoriasis. Previous studies have demonstrated the pharmacologic properties of DMF; however the properties of MEF have been under explored. Characterizing the pharmacodynamic properties of DMF and MEF will provide important insights into the mechanisms of action for delayed-release DMF versus combination products containing DMF and MEF salts. DESIGN/METHODS: DMF and MEF were applied to HEK293 cells expressing Keap1. Keap1 was immunopurified, digested and resultant peptides were analyzed by mass spectrometry to identify specific peptides modified by treatment. Primary human astrocytes were treated with DMF or MEF, and cellular glutathione was measured over 24 hours and cells were also analyzed for pharmacodynamic responses by reverse transcription polymerase chain reaction. RESULTS: DMF treatment resulted in a robust modification of specific cysteine residues in Keap1, an Nrf2 chaperone protein. MEF similarly modified Keap1, but the overall degree was significantly less. DMF produced an acute concentration-dependent depletion of glutathione; however levels recovered and rose above baseline by 24 hours. MEF did not cause acute reductions in glutathione, but did produce an increase by 24 hours. Consistent with Keap1 modification, DMF produced a robust transcriptional response in treated cells as did MEF, however the responses to MEF were of a lower magnitude. CONCLUSIONS: These studies demonstrate that DMF and MEF are both pharmacologically active, but have differing degrees of activities in multiple assay formats. These differences would be expected to result in divergent effects on downstream biology. Study Supported by: Biogen Idec Inc.

The principal aim of this study was to demonstrate the optimization and fine-tuning of quantitative and nonselective analysis of O-linked glycans released from therapeutic glycoproteins. Two approaches for quantitative release of O-linked glycans were examined: ammonia-based β-elimination and hydrazinolysis deglycosylation strategies. A significant discrepancy in deglycosylation activity was observed between the ammonia-based and hydrazinolysis procedures. Specifically, the release of O-glycans from glycoproteins was approximately 20 to 30 times more efficient with hydrazine compared with ammonia-based β-elimination reagent. In addition, the ammonia-based reagent demonstrated bias in the release of particular glycan species. A robust quantitative hydrazinolysis procedure was developed for characterization of O-glycans. The method performance parameters were evaluated. It was shown that this procedure is superior for quantitative nonselective release of O-glycans. Identity confirmation and structure elucidation of O-glycans from hydrophilic interaction chromatography (HILIC) fractions was also demonstrated using linear ion trap Fourier transform mass spectrometry (LTQ FT MS) with mass accuracy below 1ppm.

A subset of the compound repository for lead identification at Biogen Idec was characterized for its chemical stability over a 3-year period. Compounds were stored at 4 degrees C as 10 mM DMSO stocks, and a small subset of compounds was stored as lyophilized dry films. Compound integrity of 470 discrete compounds (Compound Set I) and 1917 combinatorial chemistry-derived compounds (Compound Set II) was evaluated by liquid chromatography/mass spectrometry from the time of acquisition into the library collection and after 3 years of storage. Loss of compound integrity over the 3 years of storage was observed across the 2 subsets tested. Of Compound Set I, 63% of samples retained > 80% purity, whereas 57% of samples from Compound Set II had purity greater than 60%. The stability of the lyophilized samples was superior to the samples stored as DMSO solution. Although storage at 4 degrees C as DMSO solution was adequate for the majority of compounds, the authors observed and quantified the level of degradation within the compound collection. Their study provides general insight into compound storage and selection of library subsets for future lead identification activities.