Integrative Proteogenomics for Differential Expression and Splicing Variation in a DM1 Mouse Model.

Dysregulated mRNA splicing is involved in the pathogenesis of many diseases including cancer, neurodegenerative diseases, and muscular dystrophies such as myotonic dystrophy type 1 (DM1). Comprehensive assessment of dysregulated splicing on the transcriptome and proteome level has been methodologically challenging, and thus investigations have often been targeting only few genes. Here, we performed a large-scale coordinated transcriptomic and proteomic analysis to characterize a DM1 mouse model (HSALR) in comparison to wild type. Our integrative proteogenomics approach comprised gene- and splicing-level assessments for mRNAs and proteins. It recapitulated many known instances of aberrant mRNA splicing in DM1 and identified new ones. It enabled the design and targeting of splicing-specific peptides and confirmed the translation of known instances of aberrantly spliced disease-related genes (e.g., Atp2a1, Bin1, Ryr1), complemented by novel findings (Flnc and Ywhae). Comparative analysis of large-scale mRNA and protein expression data showed quantitative agreement of differentially expressed genes and splicing patterns between disease and wild type. We hence propose this work as a suitable blueprint for a robust and scalable integrative proteogenomic strategy geared toward advancing our understanding of splicing-based disorders. With such a strategy, splicing-based biomarker candidates emerge as an attractive and accessible option, as they can be efficiently asserted on the mRNA and protein level in coordinated fashion.

Siponimod (BAF312) penetrates, distributes, and acts in the central nervous system: Preclinical insights.

BACKGROUND: Siponimod (BAF312), a selective S1P1/S1P5 agonist, reduces disability progression in secondary progressive MS. Recent observations suggest it could act via S1P1/S1P5-dependent anti-inflammatory and pro-myelination effects on CNS-resident cells. OBJECTIVE: Generate preclinical evidence confirming siponimod's CNS penetration and activity. METHODS: Siponimod's CNS penetration and distribution was explored in rodents and non-human primates (NHPs) using: Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS), quantitative whole-body autoradiography (QWBA) using 14C-radiolabeled siponimod or non-invasive single-photon emission CT (SPECT) with a validated 123I-radiolabeled siponimod analog. Functional CNS activity was investigated by S1P1 receptor quantification in brain homogenates. RESULTS: In mice/rats, siponimod treatments achieved dose-dependent efficacy and dose-proportional increase in drug blood levels, with mean brain/blood drug-exposure ratio (Brain/BloodDER) of 6-7. Efficacy in rat brain tissues was revealed by a dose-dependent reduction in brain S1P1 levels. QWBA distribution analysis in rats indicated that [14C]siponimod related radioactivity could readily penetrate CNS, with particularly high uptakes in white matter of cerebellum, corpus callosum, and medulla oblongata versus lower exposures in other areas such as olfactory bulb. SPECT monitoring in NHPs revealed CNS distribution with a brain/bloodDER of ∼6, as in rodents. CONCLUSION: Findings demonstrate siponimod's CNS penetration and distribution across species, with high translational potential to human.

Identification of a potent Janus kinase 3 inhibitor with high selectivity within the Janus kinase family.

We describe a synthetic approach toward the rapid modification of phenyl-indolyl maleimides and the discovery of potent Jak3 inhibitor 1 with high selectivity within the Jak kinase family. We provide a rationale for this unprecedented selectivity based on the X-ray crystal structure of an analogue of 1 bound to the ATP-binding site of Jak3. While equally potent compared to the Pfizer pan Jak inhibitor CP-690,550 (2) in an enzymatic Jak3 assay, compound 1 was found to be 20-fold less potent in cellular assays measuring cytokine-triggered signaling through cytokine receptors containing the common γ chain (γC). Contrary to compound 1, compound 2 inhibited Jak1 in addition to Jak3. Permeability and cellular concentrations of compounds 1 and 2 were similar. As Jak3 always cooperates with Jak1 for signaling, we speculate that specific inhibition of Jak3 is not sufficient to efficiently block γC cytokine signal transduction required for strong immunosuppression.