Gene-signature-derived IC50s/EC50s reflect the potency of causative upstream targets and downstream phenotypes.

Multiplexed gene-signature-based phenotypic assays are increasingly used for the identification and profiling of small molecule-tool compounds and drugs. Here we introduce a method (provided as R-package) for the quantification of the dose-response potency of a gene-signature as EC50 and IC50 values. Two signaling pathways were used as models to validate our methods: beta-adrenergic agonistic activity on cAMP generation (dedicated dataset generated for this study) and EGFR inhibitory effect on cancer cell viability. In both cases, potencies derived from multi-gene expression data were highly correlated with orthogonal potencies derived from cAMP and cell growth readouts, and superior to potencies derived from single individual genes. Based on our results we propose gene-signature potencies as a novel valid alternative for the quantitative prioritization, optimization and development of novel drugs.

Pharmacological Characterization of a Novel 5-Hydroxybenzothiazolone-Derived ß 2-Adrenoceptor Agonist with Functional Selectivity for Anabolic Effects on Skeletal Muscle Resulting in a Wider Cardiovascular Safety Window in Preclinical Studies.

The anabolic effects of ß 2-adrenoceptor (ß 2-AR) agonists on skeletal muscle have been demonstrated in various species. However, the clinical use of ß 2-AR agonists for skeletal muscle wasting conditions has been limited by their undesired cardiovascular effects. Here, we describe the preclinical pharmacological profile of a novel 5-hydroxybenzothiazolone (5-HOB) derived ß 2-AR agonist in comparison with formoterol as a representative ß 2-AR agonist that have been well characterized. In vitro, 5-HOB has nanomolar affinity for the human ß 2-AR and selectivity over the ß 1-AR and ß 3-AR. 5-HOB also shows potent agonistic activity at the ß 2-AR in primary skeletal muscle myotubes and induces hypertrophy of skeletal muscle myotubes. Compared with formoterol, 5-HOB demonstrates comparable full-agonist activity on cAMP production in skeletal muscle cells and skeletal muscle tissue-derived membranes. In contrast, a greatly reduced intrinsic activity was determined in cardiomyocytes and cell membranes prepared from the rat heart. In addition, 5-HOB shows weak effects on chronotropy, inotropy, and vascular relaxation compared with formoterol. In vivo, 5-HOB significantly increases hind limb muscle weight in rats with attenuated effects on heart weight and ejection fraction, unlike formoterol. Furthermore, changes in cardiovascular parameters after bolus subcutaneous treatment in rats and rhesus monkeys are significantly lower with 5-HOB compared with formoterol. In conclusion, the pharmacological profile of 5-HOB indicates superior tissue selectivity compared with the conventional ß 2-AR agonist formoterol in preclinical studies and supports the notion that such tissue-selective agonists should be investigated for the safe treatment of muscle-wasting conditions without cardiovascular limiting effects.

The critical role of kinase activity of interleukin-1 receptor-associated kinase 4 in animal models of joint inflammation.

OBJECTIVE: We have previously reported that the kinase activity of interleukin-1 receptor-associated kinase 4 (IRAK-4) is important for Toll-like receptor and interleukin-1 receptor signaling in vitro. Using mice devoid of IRAK-4 kinase activity (IRAK-4 KD mice), we undertook this study to determine the importance of IRAK-4 kinase function in complex disease models of joint inflammation. METHODS: IRAK-4 KD mice were subjected to serum transfer-induced (K/BxN) arthritis, and migration of transferred spleen lymphocytes into joints and cartilage and bone degradation were assessed. T cell response in vivo was tested in antigen-induced arthritis (AIA) by measuring the T cell-dependent antigen-specific IgG production and frequency of antigen-specific T cells in the spleen and lymph nodes. T cell allogeneic response was tested in vitro by mixed lymphocyte reaction (MLR). RESULTS: Lipopolysaccharide-induced local neutrophil influx into subcutaneous air pouches was impaired in IRAK-4 KD mice. These mice were also protected from inflammation in the K/BxN and AIA models, as shown by reduced swelling of joints. Histologic analysis of joints of K/BxN serum-injected mice revealed that bone erosion, osteoclast formation, and cartilage matrix proteoglycan loss were reduced in IRAK-4 KD mice. Assessment of T cell response by MLR, by frequency of antigen-specific clones, and by production of antigen-specific IgG did not reveal substantial differences between IRAK-4 KD and wild-type mice. CONCLUSION: These results demonstrate that IRAK-4 is a key component for the development of proarthritis inflammation, but that it is not crucial for T cell activation. Therefore, the kinase function of IRAK-4 appears to be an attractive therapeutic target in chronic inflammation.

IRAK-4 kinase activity-dependent and -independent regulation of lipopolysaccharide-inducible genes.

IRAK-4 kinase inactive (IRAK-4 KD) knock-in mice display defects in TLR- and IL-1 receptor signaling and are resistant to LPS-induced shock. In the present study we examined the LPS-induced response in IRAK-4 KD mice in more detail. We show that IRAK-4 kinase activity is required for certain aspects of TLR-mediated signaling but not for others. We found that IRAK-4 KD cells displayed reduced JNK and p38 signaling, while NF-kappaB was activated to a normal level but with delayed kinetics compared to wild-type cells. TLR4-mediated IRF3 activation was intact in these cells. Comprehensive analysis of expression of LPS-inducible genes by microarray demonstrated that IRAK-4 KD cells were severely impaired in the expression of many pro-inflammatory genes, suggesting their dependence on IRAK-4 kinase activity. In contrast, the expression of a subset of LPS-induced genes of anti-viral response was not affected by IRAK-4 kinase deficiency. Additionally, we demonstrate that LPS-activated early expression and production of some cytokines, e.g., TNF-alpha, is partially induced in the absence of IRAK-4 kinase activity. This suggests that the partially unaffected TLR4-mediated signaling could still drive expression of these genes in early phases and that IRAK-4 kinase activity is important for a more sustained anti-bacterial response.

IRAK-4 kinase activity is required for interleukin-1 (IL-1) receptor- and toll-like receptor 7-mediated signaling and gene expression.

IRAK-4 is an essential component of the signal transduction complex downstream of the IL-1- and Toll-like receptors. Although regarded as the first kinase in the signaling cascade, the role of IRAK-4 kinase activity versus its scaffold function is still controversial. To investigate the role of IRAK-4 kinase function in vivo, "knock-in" mice were generated by replacing the wild type IRAK-4 gene with a mutant gene encoding kinase-deficient IRAK-4 protein (IRAK-4 KD). IRAK-4 kinase was rendered inactive by mutating the conserved lysine residues in the ATP pocket essential for coordinating ATP. Analyses of embryonic fibroblasts and macrophages obtained from IRAK-4 KD mice demonstrate lack of cellular responsiveness to stimulation with IL-1beta or a Toll-like receptor 7 (TLR7) agonist. IRAK-4 kinase deficiency prevents the recruitment of IRAK-1 to the IL-1 receptor complex and its subsequent phosphorylation and degradation. IRAK-4 KD cells are severely impaired in NFkappaB, JNK, and p38 activation in response to IL-1beta or TLR7 ligand. As a consequence, IL-1 receptor/TLR7-mediated production of cytokines and chemokines is largely absent in these cells. Additionally, microarray analysis identified IL-1beta response genes and revealed that the induction of IL-1beta-responsive mRNAs is largely ablated in IRAK-4 KD cells. In summary, our results suggest that IRAK-4 kinase activity plays a critical role in IL-1 receptor (IL-1R)/TLR7-mediated induction of inflammatory responses.