Discovery of Phospholipase D Inhibitors with Improved Drug-like Properties and Central Nervous System Penetrance.

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 models of ALS (amyotrophic lateral sclerosis) and displays modest motor benefits in an SOD1 ALS mouse model.

A Stearoyl-Coenzyme A Desaturase Inhibitor Prevents Multiple Parkinson Disease Phenotypes in α-Synuclein Mice.

Objective: Parkinson 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.

On-MALDI-Target N-Glycan Nonreductive Amination by 2-Aminobenzoic Acid.

2-Aminobenzoic acid (2-AA) is widely used as a labeling reagent to derivatize released N-glycans at their free reducing terminus by reductive amination. 2-AA-labeled glycans have increased mass spectrometric sensitivity for their identification and enable fluorescence-chromatography-based glycan quantification. Drawbacks are that the labeling process is labor intensive and time consuming.

Defining Changes in the Spatial Distribution and Composition of Brain Lipids in the Shiverer and Cuprizone Mouse Models of Myelin Disease.

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.

Systems Analysis of Transcriptomic and Proteomic Profiles Identifies Novel Regulation of Fibrotic Programs by miRNAs in Pulmonary Fibrosis Fibroblasts.

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.

Dimethyl fumarate and monoethyl fumarate exhibit differential effects on KEAP1, NRF2 activation, and glutathione depletion in vitro.

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.

Comparison of two approaches for quantitative O-linked glycan analysis used in characterization of recombinant proteins.

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.

Discovery and investigation of O-xylosylation in engineered proteins containing a (GGGGS)n linker.

Protein engineering is a powerful tool for designing or modifying therapeutic proteins for enhanced efficacy, greater safety, reduced immunogenicity, and better delivery. GGGGS [(G4S)n] linkers are commonly used when engineering a protein, because of their flexibility and resistance to proteases. However, post-translational modifications (PTMs) can occur at the Ser residue in these linkers.

Quality assessment and analysis of Biogen Idec compound library.

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.

Expression of Sonic hedgehog-Fc fusion protein in Pichia pastoris. Identification and control of post-translational, chemical, and proteolytic modifications.

We have investigated the suitability of Pichia pastoris as an expression system for the candidate therapeutic protein, Sonic hedgehog fused to an immunoglobulin Fc domain (Shh-Fc). Sonic hedgehog is a morphogen protein involved in the patterning of a wide range of tissues during animal embryogenesis. The presence of Sonic hedgehog and its receptor, Patched, in adult nervous tissue suggests possible applications for the protein in the treatment of neurodegenerative disease and injury.