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ABSTRACTRespiratory Syncytial Virus (RSV) causes lower respiratory tract infections that can be severe and sometimes fatal. The risk for severe RSV infection is highest in infants and older adults. A safe and effective RSV vaccine for older adults represents a serious unmet medical need due to higher morbidity and mortality in this age group. In this randomized, partially double-blind, placebo-controlled, phase 1 dose-escalation study, we evaluated the safety, tolerability and immunogenicity of an investigational messenger ribonucleic acid (mRNA) vaccine encoding the RSV fusion protein (F) stabilized in the prefusion conformation. The study was conducted in healthy younger adults (ages ≥18 and ≤49 years) and healthy older adults (ages ≥60 and ≤79 years). Participants received mRNA-1777 (V171) or placebo as a single intramuscular dose. For each dose level, three sentinel participants were administered open-label mRNA-1777 (V171). Seventy-two younger adults were randomized and administered 25, 100, or 200 µg mRNA-1777 (V171) or placebo, and 107 older adults were randomized and administered 25, 100, 200 or 300 µg mRNA-1777 (V171) or placebo. Primary objectives were safety and tolerability and secondary objectives included humoral and cell-mediated immunogenicity. All dose levels of mRNA-1777 (V171) were generally well tolerated and no serious adverse events related to the vaccine were reported. Immunization with mRNA-1777 (V171) elicited a humoral immune response as measured by increases in RSV neutralizing antibody titers, serum antibody titers to RSV prefusion F protein, D25 competing antibody titers to RSV prefusion F protein, and cell-mediated immune responses to RSV-F peptides.

Large molecule quantitation by LC-MS/MS commonly relies on bottom-up or so-called surrogate peptide measurements to infer the whole-molecule concentration. This can lead to questions about what is actually being measured in the assay (intact drug and/or other drug related material). An intact sequential affinity capture (ISAC) assay was developed utilizing two different immunoaffinity (IA) reagents. The reagents were selective for the heavy and light chain of a monoclonal antibody, which when used consecutively, ensures that only the intact form of the antibody is represented by the surrogate peptide. The approach provided comparable results to a traditional sandwich IA assay indicating similar capture populations. The use of an initial ISAC assessment of affinity capture purification, should add a degree of confidence in the use of a single IA-LC-MS/MS quantitation assay.

Cultured primary neurons are a well established model for the study of neuronal function in vitro. Here we demonstrated that stable isotope labeling by amino acids in cell culture (SILAC) can be applied to a differentiated, non-dividing cell type such as primary neurons, and we applied this technique to assess changes in the neuronal phosphotyrosine proteome in response to stimulation by brain-derived neurotrophic factor (BDNF), an important molecule for the development and regulation of neuronal connections. We found that 13 proteins had SILAC ratios above 1.50 or below 0.67 in phosphotyrosine immunoprecipitations comparing BDNF-treated and control samples, and an additional 18 proteins had ratios above 1.25 or below 0.80. These proteins include TrkB, the receptor tyrosine kinase for BDNF, and others such as hepatocyte growth factor-regulated tyrosine kinase substrate and signal-transducing adaptor molecule, which are proteins known to regulate intracellular trafficking of receptor tyrosine kinases. These results demonstrate that the combination of primary neuronal cell culture and SILAC can be a powerful tool for the study of the proteomes of neuronal molecular and cellular dynamics.

Cultured primary neurons are a well established model for the study of neuronal function in vitro. Here we demonstrated that stable isotope labeling by amino acids in cell culture (SILAC) can be applied to a differentiated, non-dividing cell type such as primary neurons, and we applied this technique to assess changes in the neuronal phosphotyrosine proteome in response to stimulation by brain-derived neurotrophic factor (BDNF), an important molecule for the development and regulation of neuronal connections. We found that 13 proteins had SILAC ratios above 1.50 or below 0.67 in phosphotyrosine immunoprecipitations comparing BDNF-treated and control samples, and an additional 18 proteins had ratios above 1.25 or below 0.80. These proteins include TrkB, the receptor tyrosine kinase for BDNF, and others such as hepatocyte growth factor-regulated tyrosine kinase substrate and signal-transducing adaptor molecule, which are proteins known to regulate intracellular trafficking of receptor tyrosine kinases. These results demonstrate that the combination of primary neuronal cell culture and SILAC can be a powerful tool for the study of the proteomes of neuronal molecular and cellular dynamics.

We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion.

We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion.

Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that catalyzes the post-translational symmetric dimethylation of protein substrates. PRMT5 plays a critical role in regulating biological processes including transcription, cell cycle progression, RNA splicing, and DNA repair. As such, dysregulation of PRMT5 activity is implicated in the development and progression of multiple cancers and is a target of growing clinical interest. Described herein are the structure-based drug designs, robust synthetic efforts, and lead optimization strategies toward the identification of two novel 5,5-fused bicyclic nucleoside-derived classes of potent and efficacious PRMT5 inhibitors. Utilization of compound docking and strain energy calculations inspired novel designs, and the development of flexible synthetic approaches enabled access to complex chemotypes with five contiguous stereocenters. Additional efforts in balancing bioavailability, solubility, potency, and CYP3A4 inhibition led to the identification of diverse lead compounds with favorable profiles, promising in vivoactivity, and low human dose projections.

Whereas NGF promotes neurite outgrowth, the precursor form proNGF triggers neurite retraction.