To establish the druggability of a target, genetic validation needs to be supplemented with pharmacological validation. Pharmacological studies, especially in the kinase field, are hampered by the fact that many reference inhibitors are not fully selective for one target. Fortunately, the initial trickle of selective inhibitors released in the public domain has steadily swelled into a stream. However, rationally picking the most selective tool compound out of the increasing amounts of available inhibitors has become progressively difficult due to the lack of accurate quantitative descriptors of drug selectivity. A recently published approach, termed ‘selectivity entropy’, is an improved way of expressing selectivity as a single‐value parameter and enables rank ordering of inhibitors. We provide a guide to select the best tool compounds for pharmacological validation experiments of candidate drug targets using selectivity entropy. In addition, we recommend which inhibitors to use for studying the biology of the 20 most investigated kinases that are clinically relevant: Abl (ABL1), AKT1, ALK, Aurora A/B, CDKs, MET, CSF1R (FMS), EGFR, FLT3, ERBB2 (HER2), IKBKB (IKK2), JAK2/3, JNK1/2/3 (MAPK8/9/10), MEK1/2, PLK1, PI3Ks, p38α (MAPK14), BRAF, SRC and VEGFR2 (KDR).

No abstract

Chronic activation of Wnt/ß-catenin signaling is found in a variety of human malignancies including melanoma, colorectal and hepatocellular carcinomas. Interestingly, expression of the HCMV-encoded chemokine receptor US28 in intestinal epithelial cells promotes intestinal neoplasia in transgenic mice, which is associated with increased nuclear accumulation of ß-catenin. In this study we show that this viral receptor constitutively activates ß-catenin and enhances ß-catenin-dependent transcription. Our data illustrate that this viral receptor does not activate ß-catenin via the classical Wnt/Frizzled signaling pathway. Analysis of US28 mediated signaling indicates the involvement of the Rho-Rho kinase (ROCK) pathway in the activation of ß-catenin. Moreover, cells infected with HCMV show significant increases in ß-catenin stabilization and signaling, which is mediated to a large extent by expression of US28. The modulation of the ß-catenin signal transduction pathway by a viral chemokine receptor provides alternative regulation of this pathway, with potential relevance for the development of colon cancer and virus-associated diseases.

Background: CCX-CKR is considered to be a chemokine decoy receptor that is unable to signal. Results: Chemokines induce ␤-arrestin recruitment to CCX-CKR and pertussis toxin (PTX)-dependent CRE activity. Conclusion: PTX-sensitive G proteins hinder CCX-CKR coupling to other G proteins and consequently keep receptors silent. Significance: Recruitment of ␤-arrestin to CCX-CKR requests re-evaluation of the signaling capacity of this atypical receptor.

No abstract

Chemokines comprise a family of secreted proteins that activate G protein-coupled chemokine receptors and thereby control the migration of leukocytes during inflammation or immune surveillance. The positional information required for such migratory behavior is governed by the binding of chemokines to membrane-tethered glycosaminoglycans (GAGs), which establishes a chemokine concentration gradient. An often-observed but incompletely understood behavior of chemokines is the ability of unrelated chemokines to enhance the potency with which another chemokine subtype can activate its cognate receptor. This phenomenon has been demonstrated to occur between many chemokine combinations and across several model systems, and has been dubbed “chemokine cooperativity”. Here, we have used GAG binding-deficient chemokine mutants and cell-based functional (migration) assays to demonstrate that chemokine cooperativity is caused by competitive binding of chemokines to GAGs. This mechanistic explanation of chemokine cooperativity provides insight into chemokine gradient formation in the context of inflammation, where multiple chemokines are secreted simultaneously.

Wnt/β-catenin signaling plays essential roles in embryonic development, adult stem cell maintenance, and disease. Screening of a small molecule compound library with a β-galactosidase fragment complementation assay measuring β-catenin nuclear entry revealed TAK-715 and AMG-548 as inhibitors of Wnt-3a-stimulated β-catenin signaling. TAK-715 and AMG-548 are inhibitors of p38 mitogen-activated protein kinase, which has been suggested to regulate activation of Wnt/β-catenin signaling. However, two highly selective and equally potent p38 inhibitors, VX-745 and Scio-469, did not inhibit Wnt-3a-stimulated β-catenin signaling. Profiling of TAK-715 and AMG-548 against a panel of over 200 kinases revealed cross-reactivity with casein kinase Iδ and ɛ, which are known activators of Wnt/β-catenin signaling. Our data demonstrate that this cross-reactivity accounts for the inhibition of β-catenin signaling by TAK-715 and AMG-548 and argue against a role of p38 in Wnt/β-catenin signaling.

The effect of experimental hyperthyroidism, realized by T 4 injection, on central mediators of the hypothalamo-pituitaryinterrenal axis (HPI-axis) in common carp (Cyprinus carpio L.) was studied. Our results show that hyperthyroidism evokes a marked 3 . 2-fold reduction in basal plasma cortisol levels. Corticotropin-releasing hormone-binding protein (CRH-BP) mRNA levels in the hypothalamus, measured by real-time quantitative PCR, were significantly elevated by 40%, but CRH, urotensin-I, prepro-TRH, prohormone convertase-1 (PC1), and POMC mRNA levels were unchanged. In the pituitary pars distalis, PC1, CRH receptor-1, and POMC mRNA levels were unaffected, as was ACTH content. Plasma a-MSH concentrations were significantly elevated by 30% in hyperthyroid fish, and this was reflected in PC1 and POMC mRNA levels in pituitary pars intermedia that were increased 1 . 5-and 2 . 4-fold respectively. The a-MSH content of the pars intermedia was unchanged. Hyperthyroidism has profound effects on the basal levels of a central mediator, i.e., CRH-BP, of HPI-axis function in unstressed carp in vivo, and we conclude that HPI-and hypothalamo-pituitary-thyroidaxis functions are strongly interrelated. We suggest that the changes in plasma cortisol, thyroid hormone, and a-MSH levels reflect their concerted actions on energy metabolism.