Design and Synthesis of a Novel Series of Orally Bioavailable, CNS-Penetrant, Isoform Selective Phosphoinositide 3-Kinase γ (PI3Kγ) Inhibitors with Potential for the Treatment of Multiple Sclerosis (MS).

The lipid kinase phosphoinositide 3-kinase γ (PI3Kγ) has attracted attention as a potential target to treat a variety of autoimmune disorders, including multiple sclerosis, due to its role in immune modulation and microglial activation. By minimizing the number of hydrogen bond donors while targeting a previously uncovered selectivity pocket adjacent to the ATP binding site of PI3Kγ, we discovered a series of azaisoindolinones as selective, brain penetrant inhibitors of PI3Kγ. This ultimately led to the discovery of 16, an orally bioavailable compound that showed efficacy in murine experimental autoimmune encephalomyelitis (EAE), a preclinical model of multiple sclerosis.

Concurrent differentiation of marrow stromal cells to osteogenic and vasculogenic lineages.

When rat bone marrow stromal (BMS) cells were seeded on aligned type I collagen scaffolds and cultured in osteogenic media, they underwent simultaneous maturation and differentiation into osteogenic and vascular cell lineages. In addition, these cells produced mineralized matricellular deposits. BMS cells were seeded in Petri dish or the collagen scaffold, cultured in osteogenic media for 3, 6, and 9 d and subsequently processed for immunohistochemical and cytochemical analysis. Immunolocalization of lineage-specific proteins were visualized using confocal microscopy and mRNA transcript analysis was performed by real-time quantitative polymerase chain reaction (RT-qPCR). The alkaline phosphatase activity and calcium content significantly increased over the observed period of time in an osteogenic medium. Sheets of abundant Pecam (CD31), Flk-1 (VEGFR-2), tomato lectin (TL/LEL), and alpha-smooth muscle actin (alpha-SMA) positive cells were observed in the collagen scaffolds. Nascent capillary-like vessels were also seen amidst the osteoblasts in osteogenic culture, augmenting the maturation and differentiation of BMS cells into osteoblasts. In our in vitro study, concurrent differentiation of BMS cells, a heterogeneous cell population with multilineage differentiation potential, to osteogenic and vascular lineages demonstrated that the substrates (three-dimensional (3-D), collagen type I, aligned fibrils) had a profound effect on guiding the differentiation pathway of BMS cells.

Requirement of Staphylococcus aureus ATP-binding cassette-ATPase FhuC for iron-restricted growth and evidence that it functions with more than one iron transporter.

In Staphylococcus aureus, fhuCBG encodes an ATP-binding cassette (ABC) transporter that is required for the transport of iron(III)-hydroxamates; mutation of either fhuB or fhuG eliminates transport. In this paper, we describe construction and characterization of an S. aureus fhuCBG deletion strain. The delta fhuCBG::ermC mutation not only resulted in a strain that was incapable of growth on iron(III)-hydroxamates as a sole source of iron but also resulted in a strain which had a profound growth defect in iron-restricted laboratory media. The growth defect was not a result of the inability to transport iron(III)-hydroxamates since S. aureus fhuG::Tn917 and S. aureus fhuD1::Km fhuD2::Tet mutants, which are also unable to transport iron(III)-hydroxamates, do not have similar iron-restricted growth defects. Complementation experiments demonstrated that the growth defect of the delta fhuCBG::ermC mutant was the result of the inability to express FhuC and that this was the result of an inability to transport iron complexed to the S. aureus siderophore staphylobactin. Transport of iron(III)-staphylobactin is dependent upon SirA (binding protein), SirB (permease), and SirC (permease). S. aureus expressing FhuC with a Walker A K42N mutation could not utilize iron(III)-hydroxamates or iron(III)-staphylobactin as a sole source of iron, supporting the conclusion that FhuC, as expected, functions with FhuB, FhuG, and FhuD1 or FhuD2 to transport iron(III)-hydroxamates and is the "genetically unlinked" ABC-ATPase that functions with SirA, SirB, and SirC to transport iron(III)-staphylobactin. Finally, we demonstrated that the delta fhuCBG::ermC strain had decreased virulence in a murine kidney abscess model.