1,2,3-Triazole-totarol conjugates as potent PIP5K1α lipid kinase inhibitors.

The human phosphatidylinositol 4-phosphate 5-kinase type I α (hPIP5K1α) plays a key role in the development of prostate cancer. In this work, seventeen derivatives of the natural diterpene totarol were prepared by copper(I)-catalysed Huisgen 1,3-dipolar cycloaddition reaction of the correspondingO-propargylated totarol with aryl or alkyl azides and screened for their inhibitory activities toward hPIP5K1α. Five compounds, 3a, 3e, 3f, 3i, and 3r, strongly inhibited the enzyme activity with IC50 values of 1.44, 0.46, 1.02, 0.79, and 3.65 µM, respectively, with the most potent inhibitor 3e 13-[(1-(3-nitrophenyl)triazol-4yl)methoxy]-totara-8,11,13-triene). These compounds were evaluated on their antiproliferative effects in a panel of prostate cancer cell lines. Compound 3r inhibited the proliferation of LNCaP, PC3 and DU145 cells at 20 µM, strongly, but also has strong cytotoxic effects on all tested cells.

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.

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.

Highly 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-tetrahydroindeno[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 with 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, provides 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.

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.

[2.2]Paracyclophane-Based TCN-201 Analogs as GluN2A-Selective NMDA Receptor Antagonists.

Recent studies have shown the involvement of GluN2A subunit-containing NMDA receptors in various neurological and pathological disorders. In the X-ray crystal structure, TCN-201 (1) and analogous pyrazine derivatives 2 and 3 adopt a U-shape (hairpin) conformation within the binding site formed by the ligand binding domains of the GluN1 and GluN2A subunits. In order to mimic the resulting π/π-interactions of two aromatic rings in the binding site, a [2.2]paracyclophane system was designed to lock these aromatic rings in a parallel orientation. Acylation of [2.2]paracyclophane (5) with oxalyl chloride and chloroacetyl chloride and subsequent transformations led to the oxalamide 7, triazole 10 and benzamides 12. The GluN2A inhibitory activities of the paracyclophane derivatives were tested with two-electrode voltage clamp electrophysiology using Xenopus laevis oocytes expressing selectively functional NMDA receptors with GluN2A subunit. The o-iodobenzamide 12 b with the highest similarity to TCN-201 showed the highest GuN2A inhibitory activity of this series of compounds. At a concentration of 10 µM, 12 b reached 36 % of the inhibitory activity of TCN-201 (1). This result indicates that the [2.2]paracyclophane system is well accepted by the TCN-201 binding site.

Development of an in vitro screening assay for PIP5K1α lipid kinase and identification of potent inhibitors.

The human phosphatidylinositol 4-phosphate 5-kinase type I α (hPIP5K1α) participates in the phosphoinositide-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway. Despite the evidence that hPIP5K1α plays a role in the development of prostate cancer (PCa), only one inhibitor is known to date. With the aim of identifying new inhibitors, a nonradiometric assay for measurement of the hPIP5K1α enzyme activity was developed. The assay is based on the separation of the fluorescently labeled substrate phosphatidylinositol-4-phosphate (PI(4)P) and the resulting product phosphatidylinositol-4,5-bisphosphate (PIP2 ) by capillary electrophoresis (CE). Furthermore, an inactive mutant K261A of hPIP5K1α was generated by site-directed mutagenesis and used as a control. Michaelis-Menten analysis revealed a Km value of 21.6 µm and Vmax of 0.65 pmol·min-1 for the cosubstrate ATP. The average Z' value was determined to be 0.86, indicating a high reliability of the assay. An in silico screening of an in-house compound library was performed employing the crystal structure of zebrafish PIP5K1α. By applying this strategy, three compounds with a 2-amino-3-cyano-4H-pyranobenzoquinone scaffold were identified and tested using the CE-based assay. These compounds inhibited hPIP5K1α to > 90% at a concentration of 50 µm. Subsequently, the inhibitory activity of all compounds with a pyranobenzoquinone scaffold (29) was tested on hPIP5K1α. Compound 4-(2-amino-3-cyano-6-hydroxy-5,8-dioxo-7-undecyl-5,8-dihydro-4H-chromen-4-yl)benzoic acid appeared to be the most potent inhibitor of hPIP5K1α identified so far with an IC50 value of 1.55 µm, exhibiting a substrate-competitive mode of action. The effects of this compound on cell viability and the induction of apoptosis were investigated in LNCaP, DU145, and PC3 PCa cells.

Direct, gabapentin-insensitive interaction of a soluble form of the calcium channel subunit α2δ-1 with thrombospondin-4.

The α2δ-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 α2δ-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/α2δ-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 α2δ-1 (α2δ-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 α2δ-1 protein. These results provide biochemical evidence that supports a specific role of TSP-4 among the TSPs in mediating the binding to neuronal α2δ-1 and suggest that gabapentin does not directly target TSP/α2δ-1 interaction to alleviate neuropathic pain.

Involvement of glutamate, oxidative stress and inducible nitric oxide synthase in the convulsant activity of ciprofloxacin in mice.

This study investigated the potential convulsive activity of ciprofloxacin in mice and the possible mechanism(s) of this activity. Intraperitoneal (i.p.) administration of ciprofloxacin into mice resulted in convulsive seizures in a dose-dependent manner. The clonic median convulsant dose (CD(50)) of ciprofloxacin in mice was increased by pretreatment with dizocilpine, alpha-lipoic acid or aminoguanidine, not changed by pretreatment with 7-nitroindazole and decreased by pretreatment with L-arginine and fenbufen. The increase in nitric oxide (NO) production and malondialdehyde (MDA) level as well as the decrease in intracellular reduced glutathione (GSH) level and glutathione peroxidase (GSH-Px) activity induced by the estimated clonic CD(50) of ciprofloxacin in mice brain was inhibited by pretreatment with dizocilpine, alpha-lipoic acid or aminoguanidine. These biochemical alterations were not changed by pretreatment with 7-nitroindazole but enhanced by pretreatment with L-arginine. The elevation induced by the clonic CD(50) of ciprofloxacin in brain glutamate level was not changed by pretreatment with MK-801, alpha-lipoic acid, aminoguanidine or L-arginine. Combined treatment of mice with fenbufen and ciprofloxacin produced elevation of brain NO production and glutamate and MDA levels as well as inhibition of brain intracellular GSH level and GSH-Px activity. In addition, i.p. administration of the clonic CD(50) of ciprofloxacin produced an increase in inducible but not in neuronal NO synthase mRNA and protein expressions in mice brain. These results suggest that elevation of brain glutamate levels with consequent oxidative stress and increase in the expression and activity of brain inducible NO synthase may play a pivotal role in ciprofloxacin-induced convulsive seizures.