Discovery of Highly Isoform Selective Orally Bioavailable Phosphoinositide 3-Kinase (PI3K)-γ Inhibitors.

In this paper, we describe the discovery and optimization of a new chemotype of isoform selective PI3Kγ inhibitors. Starting from an HTS hit, potency and physicochemical properties could be improved to give compounds such as 15, which is a potent and remarkably selective PI3Kγ inhibitor with ADME properties suitable for oral administration. Compound 15 was advanced into in vivo studies showing dose-dependent inhibition of LPS-induced airway neutrophilia in rats when administered orally.

Comparing Flow Cytometry QBeads PlexScreen Assays with Other Immunoassays for Determining Multiple Analytes.

Immunoassays, utilizing the affinity of antibodies to their antigens, are powerful techniques and have been widely used for quantifying analytes, such as cytokines, in biological samples in the clinic and in drug discovery. Various immunoassays have been developed to fit for different purposes. Recently, bead-based flow cytometry assays have emerged as interesting options for multiplex quantification of analytes. In this study, we compared high-throughput flow cytometry multiplex iQue QBeads PlexScreen assays with several other commonly used immunoassays, including MSD, Luminex, ELISA, HTRF, and AlphaLISA assays. Head-to-head comparisons of quantification data of the following cytokines were made: (1) IL-2, IL-4, IL-6, IL-13, IL-17A, IFNγ, KC/GRO, RANTES, and TNFα in mouse bronchoalveolar lavage fluid samples; (2) IL-10 and TNFα in supernatants from a THP-1 cell assay; (3) IL-6, IL-10, IL-12p70, and TNFα in supernatants from a human monocyte-derived dendritic cell assay; and (4) IL-2 in supernatants from a human CD4+ cell assay. The results demonstrated a good assay correlation between the iQue and the compared assays for the cytokine studied. Although overall good assay correlations were observed, our results showed that the iQue assay generated different absolute cytokine values for some cytokines in the same sample sets compared with other assays.

Design and Synthesis of Soluble and Cell-Permeable PI3Kδ Inhibitors for Long-Acting Inhaled Administration.

PI3Kδ is a lipid kinase that is believed to be important in the migration and activation of cells of the immune system. Inhibition is hypothesized to provide a powerful yet selective immunomodulatory effect that may be beneficial for the treatment of conditions such as asthma or rheumatoid arthritis. In this work, we describe the identification of inhibitors based on a thiazolopyridone core structure and their subsequent optimization for inhalation. The initially identified compound (13) had good potency and isoform selectivity but was not suitable for inhalation. Addition of basic substituents to a region of the molecule pointing to solvent was tolerated (enzyme inhibition pIC50 > 9), and by careful manipulation of the pKa and lipophilicity, we were able to discover compounds (20b, 20f) with good lung retention and cell potency that could be taken forward to in vivo studies where significant target engagement could be demonstrated.

Adiponectin receptor 2 deficiency results in reduced atherosclerosis in the brachiocephalic artery in apolipoprotein E deficient mice.

Adiponectin has been shown to have beneficial cardiovascular effects and to signal through the adiponectin receptors, AdipoR1 and AdipoR2. The original aim of this study was to investigate the effect of combined AdipoR1 and AdipoR2 deficiency (AdipoR1(-/-)AdipoR2(-/-)) on atherosclerosis. However, we made the interesting observation that AdipoR1(-/-) AdipoR2(-/-) leads to embryonic lethality demonstrating the critical importance of the adiponectin signalling system during development. We then investigated the effect of AdipoR2-ablation on the progression of atherosclerosis in apolipoprotein E deficient (ApoE(-/-)) mice. AdipoR2(-/-)ApoE(-/-) mice fed an atherogenic diet had decreased plaque area in the brachiocephalic artery compared with AdipoR2(+/+) ApoE(-/-) littermate controls as visualized in vivo using an ultrasound biomicroscope and confirmed by histological analyses. The decreased plaque area in the brachiocephalic artery could not be explained by plasma cholesterol levels or inflammatory status. However, accumulation of neutral lipids was decreased in peritoneal macrophages from AdipoR2(-/-)ApoE(-/-) mice after incubation with oxidized LDL. This effect was associated with lower CD36 and higher ABCA1 mRNA levels in peritoneal macrophages from AdipoR2(-/-)ApoE(-/-) mice compared with AdipoR2(+/+)ApoE(-/-) controls after incubation with oxidized LDL. In summary, we show that adiponectin receptors are crucial during embryonic development and that AdipoR2-deficiency slows down the progression of atherosclerosis in the brachiocephalic artery of ApoE-deficient mice.

Transcriptional control of SLC26A4 is involved in Pendred syndrome and nonsyndromic enlargement of vestibular aqueduct (DFNB4).

Although recessive mutations in the anion transporter gene SLC26A4 are known to be responsible for Pendred syndrome (PS) and nonsyndromic hearing loss associated with enlarged vestibular aqueduct (EVA), also known as "DFNB4," a large percentage of patients with this phenotype lack mutations in the SLC26A4 coding region in one or both alleles. We have identified and characterized a key transcriptional regulatory element in the SLC26A4 promoter that binds FOXI1, a transcriptional activator of SLC26A4. In nine patients with PS or nonsyndromic EVA, a novel c.-103T-->C mutation in this regulatory element interferes with FOXI1 binding and completely abolishes FOXI1-mediated transcriptional activation. We have also identified six patients with mutations in FOXI1 that compromise its ability to activate SLC26A4 transcription. In one family, the EVA phenotype segregates in a double-heterozygous mode in the affected individual who carries single mutations in both SLC26A4 and FOXI1. This finding is consistent with our observation that EVA occurs in the Slc26a4(+/-); Foxi1(+/-) double-heterozygous mouse mutant. These results support a novel dosage-dependent model for the molecular pathogenesis of PS and nonsyndromic EVA that involves SLC26A4 and its transcriptional regulatory machinery.

Eya1 regulates the growth of otic epithelium and interacts with Pax2 during the development of all sensory areas in the inner ear.

Members of the Eyes absent (Eya) gene family are important for auditory system development. While mutations in human EYA4 cause late-onset deafness at the DFNA10 locus, mutations in human EYA1 cause branchio-oto-renal (BOR) syndrome. Inactivation of Eya1 in mice causes an early arrest of the inner ear development at the otocyst stage. To better understand the role of Eya1 in inner ear development, we analyzed the cellular and molecular basis of the early defect observed in the Eya1 mutant embryos. We report here that Eya1-/- otic epithelium shows reduced cell proliferation from E8.5 and increased cell apoptosis from E9.0, thus providing insights into the cellular basis of inner ear defect which occurred in the absence of Eya1. Previous studies have suggested that Pax, Eya and Six genes function in a parallel or independent pathway during inner ear development. However, it remains unknown whether Pax genes interact with Eya1 or Six1 during inner ear morphogenesis. To further evaluate whether Pax genes function in the Eya1-Six1 pathway or whether they interact with Eya1 or Six1 during inner ear morphogenesis, we have analyzed the expression pattern of Eya1, Pax2 and Pax8 on adjacent sections of otic epithelium from E8.5 to 9.5 by in situ hybridization and the inner ear gross structures of Pax2, Eya1 and Six1 compound mutants at E17.5 by latex paintfilling. Our data strongly suggest that Pax2 interacts with Eya1 during inner ear morphogenesis, and this interaction is critical for the development of all sensory areas in the inner ear. Furthermore, otic marker analysis in both Eya1-/- and Pax2-/- embryos indicates that Eya1 but not Pax2 regulates the establishment of regional specification of the otic vesicle. Together, these results show that, while Eya1 exerts an early function essential for normal growth and patterning of the otic epithelium, it also functionally synergizes with Pax2 during the morphogenesis of all sensory areas of mammalian inner ear.