We also tested the consequences of two widely used NFB inhibitors, TPCK (tosyl-L-phenylalanyl-chloromethane ketone) that affects NFB at concentrations 10 M by inhibiting IKK [21] (Physique 2D) and proteasome inhibitor bortezomib active in sub-micromolar range (Physique 2E). a distinct role for other CDKs in the NFB pathway. 6H05 (trifluoroacetate salt) We used this assay to characterize a series of thienopyridines with in vitro bone anabolic activity, one of which was identified as a selective CDK8/19 inhibitor. Thienopyridines inhibited luciferase induction in the WT but not dKO cells and their IC50 values in the WT reporter assay showed near-perfect correlation (R2 = 0.98) with their reported activities in a bone anabolic activity assay, confirming that this latter function is mediated by CDK8/19 and validating our assay as a robust and quantitative method for CDK8/19 inhibition. = 3). Asterisks: 0.01 (= 3). Asterisks: 0.01 for the difference between TNF and TNF + senexin B readouts. (C) Effects of different concentrations of senexin B on luciferase expression in the indicated WT and dKO 293 clones treated with 10 ng/mL TNF for 3 h. % control (Y axis) was calculated relative to cells without the inhibitor. (DCF) Effects of different concentrations of dCA, TPCK and bortezomib on luciferase expression in 293-WT-NFKB-LUC#8 and 293-dKO-NFKB-LUC#2 reporter clones treated with 10 ng/mL TNF for 3 h. Physique 2D shows the effects of another, more potent CDK8/19 inhibitor, dCA (didehydro-Cortistatin A), an equipotent analog of cortistatin A [20] on TNF-induced luciferase activity in these reporter cell lines. dCA had no effect on reporter induction in dKO cells but suppressed such induction in the WT reporter with IC50 of 1 1.3 nM (as compared to 114 nM for senexin B in the same cells). Maximal inhibition of the reporter induction by dCA reached a plateau 6H05 (trifluoroacetate salt) at ~80%, similar to the maximal effect of senexin B. We also tested Rabbit Polyclonal to NMU the effects of two widely used NFB inhibitors, TPCK (tosyl-L-phenylalanyl-chloromethane ketone) that affects NFB at concentrations 10 M by inhibiting IKK [21] (Physique 2D) and proteasome inhibitor bortezomib active in sub-micromolar range (Physique 2E). Both TPCK and bortezomib inhibited the reporter activity in both WT and dKO with comparable IC50 values, with the highest concentrations of TPCK achieving complete suppression of NFB. 3.3. Effects of Inhibitors of Other CDKs in the NFB-Dependent Cell-Based Assay We further tested several inhibitors of other CDKs in the same assay. Flavopiridol (Alvociclib) is usually a potent inhibitor of multiple CDKs with preferential activity against CDK9, CDK4 and CDK7 [22]. Dinaciclib selectively inhibits cyclin dependent kinases CDK1, CDK2, CDK5 and CDK9 [23]. THZ1 inhibits CDK7, CDK12 and CDK13 [24] and palbociclib selectively inhibits CDK4 and CDK6 [25]. Flavopiridol, dinaciclib and THZ1 all completely inhibited NFB-dependent 6H05 (trifluoroacetate salt) promoter activation with indistinguishable IC50 values in WT and dKO cells, without the plateau common for CDK8/19 inhibitors. In contrast, Palbociclib showed only weak inhibitory effects at high concentrations ( 1 M), in both WT and dKO cells (Physique 3). Open in a separate window Physique 3 Effects of flavopiridol, dinaciclib, THZ1 and palbociclib at different concentrations around the induced NFB reporter activity in WT and dKO 293 cells treated with 10 ng/mL TNF for 3 h. 3.4. Analysis of a Series of Thienopyridine-Derivatives with Bone Anabolic Activity A recent publication reported that a thienopyridine derivative (15w) is usually a selective CDK8/19 inhibitor that (along with senexin B) promotes osteoblast mineralization and bone regeneration [17]. 15w is usually one of a series of compounds that were originally discovered and optimized for in vitro bone anabolic activity using an alkaline phosphatase (ALPase) activity assay in a mouse bone marrow stromal cell line ST2 [16]. To test if the activity of other compounds in the ALPase assay was associated with CDK8/19 inhibition, six thienopyridines with different ALPase-enhancing activities.
