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The methylation status of a 4.4-kb 5' end of the myelin-associated glycoprotein (MAG) gene was assessed in cells with different levels of transcriptional activity of the gene, i.e., liver, brain, O-2A oligodendrocyte precursors cells, mature oligodendrocytes, and glioma C6 cells. Purified DNA was digested with methylation-sensitive restriction enzymes, and the cuts were mapped by the indirect end-labeling technique. The restriction sites within the 4.4-kb fragment revealed a highly heterogenous methylation pattern among cells and tissues, and liver DNA was the most heavily methylated. Most of the restriction sites were partly demethylated in the nervous system cells. Notably, two adjacent Hha1 sites at +94 and +96 were fully methylated in liver, but partially demethylated in the brain, OL, and O2A. Two Hpa2 site located at -1836 and at -39 were progressively demethylated in oligodendrocyte lineage cells, indicating specific hypomethylation associated with the oligodendrocytic differentiation. Most of the restriction sites were weakly methylated in the DNA from neoplastic C6 cells, although the Hha1 sites were fully methylated. No clear-cut correlation between the extent of CpG dinucleotide methylation and the chromatin conformation was found. For example, out of four heavily methylated sites only two comapped with MNase hypersensitive sites. Also, the -1836 Hpa2 site whose demethylation is concomitant with oligodendrocytic differentiation seems to be localized within precisely positioned nucleosomal arrays of the MAG gene chromatin. The results indicate that the MAG gene undergoes progressive demethylation concomitant with the oligodendrocyte differentiation/maturation. However, certain CpG dinucleotides remain heavily methylated even in the fully active gene in mature oligodendrocytes, indicating that they may be essential in maintaining proper chromatin structure.

Aberrant activation of the Wnt pathway is believed to drive the development and growth of some cancers. The central role of CK1γ in Wnt signal transduction makes it an attractive target for the treatment of Wnt-pathway dependent cancers. We describe a structure-based approach that led to the discovery of a series of pyridyl pyrrolopyridinones as potent and selective CK1γ inhibitors. These compounds exhibited good enzyme and cell potency, as well as selectivity against other CK1 isoforms. A single oral dose of compound 13 resulted in significant inhibition of LRP6 phosphorylation in a mouse tumor PD model.

Recapitulating human disease pathophysiology using genetic animal models is a powerful approach to enable mechanistic understanding of genotype-phenotype relationships for drug development. Na V 1.7 is a sodium channel expressed in the peripheral nervous system with strong human genetic validation as a pain target. Efforts to identify novel analgesics that are nonaddictive resulted in industry exploration of a class of sulfonamide compounds that bind to the fourth voltage-sensor domain of Na V 1.7. Due to sequence differences in this region, sulfonamide blockers generally are potent on human but not rat Na V 1.7 channels. To test sulfonamide-based chemical matter in rat models of pain, we generated a humanized Na V 1.7 rat expressing a chimeric Na V 1.7 protein containing the sulfonamide-binding site of the human gene sequence as a replacement for the equivalent rat sequence. Unexpectedly, upon transcription, the human insert was spliced out, resulting in a premature stop codon. Using a validated antibody, Na V 1.7 protein was confirmed to be lost in the brainstem, dorsal root ganglia, sciatic nerve, and gastrointestinal tissue but not in nasal turbinates or olfactory bulb in rats homozygous for the knock-in allele (HOM-KI). HOM-KI rats exhibited normal intraepidermal nerve fiber density with reduced tetrodotoxin-sensitive current density and action potential firing in small diameter dorsal root ganglia neurons. HOM-KI rats did not exhibit nociceptive pain responses in hot plate or capsaicin-induced flinching assays and did not exhibit neuropathic pain responses following spinal nerve ligation. Consistent with expression of chimeric Na V 1.7 in olfactory tissue, HOM-KI rats retained olfactory function. This new genetic model highlights the necessity of Na V 1.7 for pain behavior in rats and indicates that sufficient inhibition of Na V 1.7 in humans may reduce pain in neuropathic conditions. Due to preserved olfactory function, this rat model represents an alternative to global Na V 1.7 knockout mice that require time-intensive hand feeding during early postnatal development.