Broad-Spectrum Anticancer Activity and Pharmacokinetic Properties of a Prenyloxy-Substituted Indeno[1,2-b]indole Derivative, Discovered as CK2 Inhibitor

Protein kinase CK2 is involved in regulating cellular processes, such as cell cycle, proliferation, migration, and apoptosis, making it an attractive anticancer target. We previously described a prenyloxy-substituted indeno[1,2-b]indole (5-isopropyl-4-(3-methylbut-2-enyloxy)-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (4p)) as a very potent inhibitor of CK2 holoenzyme (IC50 = 25 nM). Here, we report the broad-spectrum anticancer activity of 4p and provide substantial progress on its pharmacokinetic properties. Using a cell-based CK2 activity assay and live-cell imaging of cultured A431, A549, and LNCaP cancer cell lines, cellular CK2 target engagement was shown as well as strong antiproliferative, anti-migratory and apoptosis-inducing effects of 4p. Furthermore, evidence was found for the ability of 4p to disrupt A549 spheroid cohesion. A series of LC-MS/MS experiments revealed high and rapid cellular uptake (intracellular concentration is approximately 5 µM after 1 h incubation) and low metabolic stability of 4p. These results point to the value of 4p as a potent CK2 inhibitor with promising anticancer activities and should trigger future medicinal chemistry efforts to improve the drug-like properties of this compound

4,5,7‐trisubstituted indeno[1,2‐ b ]indole inhibits CK2 activity in tumor cells equivalent to CX‐4945 and shows strong anti‐migratory effects

International audienceHighly pleiotropic and constitutively active protein kinase CK2 is a key target in cancer therapy, but only one small-molecule inhibitor has reached clinical trials - CX-4945. In this study we present the indeno[1,2-b]indole derivative 5-isopropyl-4-methoxy-7-methyl-5,6,7,8-tetrahydro-indeno[1,2-b]indole-9,10-dione (5a-2) that decreased the intracellular CK2 activity in A431, A549 and LNCaP tumor cell lines analogous to CX-4945 (>75% inhibition at 20 µM) and similarly blocked CK2-specific Akt phosphorylation in LNCaP cells. Cellular uptake analysis demonstrated higher intracellular concentrations of 5a-2 (408.3 nM) compared to CX-4945 (119.3 nM). This finding clarifies the comparable effects of both compounds on the intracellular CK2 activity despite their different inhibitory potency in vitro [IC50 = 25 nM (5a-2) and 3.7 nM (CX-4945)]. Examination of the effects of both CK2 inhibitors on cancer cells using live cell imaging revealed notable differences. Whereas CX-4945 showed a stronger pro-apoptotic effect on tumor cells, 5a-2 was more effective in inhibiting tumor cell migration. Our results showed that 49% of intracellular CX-4945 was localized in the nuclear fraction, whereas 71% of 5a-2 was detectable in the cytoplasm. The different subcellular distribution, and thus the site of CK2 inhibition, provide a possible explanation for the different cellular effects. Our study indicates that investigating CK2-inhibition-mediated cellular effects in relation to the subcellular sites of CK2 inhibition may help to improve our understanding of the preferential roles of CK2 within different cancer cell compartments

Direct, gabapentin-insensitive interaction of a soluble form of the calcium channel subunit alpha(2)delta-1 with thrombospondin-4

The alpha(2)delta-1 subunit of voltage-gated calcium channels binds to gabapentin and pregabalin, mediating the analgesic action of these drugs against neuropathic pain. Extracellular matrix proteins from the thrombospondin (TSP) family have been identified as ligands of alpha(2)delta-1 in the CNS. This interaction was found to be crucial for excitatory synaptogenesis and neuronal sensitisation which in turn can be inhibited by gabapentin, suggesting a potential role in the pathogenesis of neuropathic pain. Here, we provide information on the biochemical properties of the direct TSP/alpha(2)delta-1 interaction using an ELISA-style ligand binding assay. Our data reveal that full-length pentameric TSP-4, but neither TSP-5/COMP of the pentamer-forming subgroup B nor TSP-2 of the trimer-forming subgroup A directly interact with a soluble variant of alpha(2)delta-1 (alpha(2)delta-1S). Interestingly, this interaction is not inhibited by gabapentin on a molecular level and is not detectable on the surface of HEK293-EBNA cells over-expressing alpha(2)delta-1 protein. These results provide biochemical evidence that supports a specific role of TSP-4 among the TSPs in mediating the binding to neuronal a2d-1 and suggest that gabapentin does not directly target TSP/alpha(2)delta-1 interaction to alleviate neuropathic pain

Angiotensin II type 1 receptor localizes at the blood-bile barrier in humans and pigs

Animal models and clinical studies suggest an influence of angiotensin II (AngII) on the pathogenesis of liver diseases via the renin-angiotensin system. AngII application increases portal blood pressure, reduces bile flow, and increases permeability of liver tight junctions. Establishing the subcellular localization of angiotensin II receptor type 1 (AT1R), the main AngII receptor, helps to understand the effects of AngII on the liver. We localized AT1R in situ in human and porcine liver and porcine gallbladder by immunohistochemistry. In order to do so, we characterized commercial anti-AT1R antibodies regarding their capability to recognize heterologous human AT1R in immunocytochemistry and on western blots, and to detect AT1R using overlap studies and AT1R-specific blocking peptides. In hepatocytes and canals of Hering, AT1R displayed a tram-track-like distribution, while in cholangiocytes AT1R appeared in a honeycomb-like pattern; i.e., in liver epithelia, AT1R showed an equivalent distribution to that in the apical junctional network, which seals bile canaliculi and bile ducts along the blood-bile barrier. In intrahepatic blood vessels, AT1R was most prominent in the tunica media. We confirmed AT1R localization in situ to the plasma membrane domain, particularly between tight and adherens junctions in both human and porcine hepatocytes, cholangiocytes, and gallbladder epithelial cells using different anti-AT1R antibodies. Localization of AT1R at the junctional complex could explain previously reported AngII effects and predestines AT1R as a transmitter of tight junction permeability