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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 (IC 50 = 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.

Since the approval of imatinib in 2001, kinase inhibitors have revolutionized cancer therapies. Inside this family of phosphotransferases, casein kinase 2 (CK2) is of great interest and numerous scaffolds have been investigated to design CK2 inhibitors. Recently, functionalized indeno[1,2- b ]indoles have been revealed to have high potency against human cancer cell lines such as MCF-7 breast carcinoma and A-427 lung carcinoma. 4-Methoxy-5-isopropyl-5,6,7,8-tetrahydroindeno[1,2- b ]indole-9,10-dione (THN7), identified as a potent inhibitor of CK2 (IC 50 = 71 nM), was selected for an encapsulation study in order to evaluate its antiproliferative activity as THN7-loaded cyclodextrin nanoparticles. Four α-cyclodextrins (α-CDs) were selected to encapsulate THN7 and all experiments indicated that the nanoencapsulation of this CK2 inhibitor in α-CDs was successful. No additional surface-active agent was used during the nanoformulation process. Nanoparticles formed between THN7 and α-C₆H 13 amphiphilic derivative gave the best results in terms of encapsulation rate (% of associated drug = 35%), with a stability constant (K 11 ) of 298 mol·L -1 and a size of 132 nm. Hemolytic activity of the four α-CDs was determined before the in cellulo evaluation and the α-C₆H 13 derivative gave the lowest value of hemolytic potency (HC 50 = 1.93 mol·L -1 ). Only the THN7-loaded cyclodextrin nanoparticles showing less toxicity on human erythrocytes (α-C₆H 13 , α-C₈H 17 and α-C₄H₉) were tested against A-427 cells. All drug-loaded nanoparticles caused more cytotoxicity against A-427 cells than THN7 alone. Based on these results, the use of amphiphilic CD nanoparticles could be considered as a drug delivery system for indeno[1,2- b ]indoles, allowing an optimized bioavailability and offering perspectives for the in vivo development of CK2 inhibitors., Competing Interests: The authors declare no conflict of interest.

Protein kinase CK2, a member of the eukaryotic protein kinase superfamily, is associated with cancer and other human pathologies and thus an attractive drug target. The indeno[1,2- b ]indole scaffold is a novel lead structure to develop ATP-competitive CK2 inhibitors. Some indeno[1,2- b ]indole-based CK2 inhibitors additionally obstruct ABCG2, an ABC half transporter overexpressed in breast cancer and co-responsible for drug efflux and resistance. Comprehensive derivatization studies revealed substitutions of the indeno[1,2- b ]indole framework that boost either the CK2 or the ABCG2 selectivity or even support the dual inhibition potential. The best indeno[1,2- b ]indole-based CK2 inhibitor described yet (IC 50 = 25 nM) is 5-isopropyl-4-(3-methylbut-2-enyl-oxy)-5,6,7,8-tetrahydroindeno[1,2- b ]indole-9,10-dione ( 4p ). Herein, we demonstrate the membrane permeability of 4p and describe co-crystal structures of 4p with CK2α and CK2α', the paralogs of human CK2 catalytic subunit. As expected, 4p occupies the narrow, hydrophobic ATP site of CK2α/CK2α', but surprisingly with a unique orientation: its hydrophobic substituents point towards the solvent while its two oxo groups are hydro-gen-bonded to a hidden water molecule. An equivalent water molecule was found in many CK2α structures, but never as a critical mediator of ligand binding. This unexpected binding mode is independent of the interdomain hinge/helix αD region conformation and of the salt content in the crystallization medium., Competing Interests: The authors declare no conflict of interest.

Protein kinase CK2, initially designated as casein kinase 2, is an ubiquitously expressed serine/threonine kinase. This enzyme, implicated in many cellular processes, is highly expressed and active in many tumor cells. A large number of compounds has been developed as inhibitors comprising different backbones. Beside others, structures with an indeno[1,2- b ]indole scaffold turned out to be potent new leads. With the aim of developing new inhibitors of human protein kinase CK2, we report here on the generation of common feature pharmacophore model to further explain the binding requirements for human CK2 inhibitors. Nine common chemical features of indeno[1,2- b ]indole-type CK2 inhibitors were determined using MOE software (Chemical Computing Group, Montreal, Canada). This pharmacophore model was used for database mining with the aim to identify novel scaffolds for developing new potent and selective CK2 inhibitors. Using this strategy several structures were selected by searching inside the ZINC compound database. One of the selected compounds was bikaverin (6,11-dihydroxy-3,8-dimethoxy-1-methylbenzo[ b ]xanthene-7,10,12-trione), a natural compound which is produced by several kinds of fungi. This compound was tested on human recombinant CK2 and turned out to be an active inhibitor with an IC 50 value of 1.24 µM.

Due to their system of annulated 6-5-5-6-membered rings, indenoindoles have sparked great interest for the design of ATP-competitive inhibitors of human CK2. In the present study, we prepared twenty-one indeno[1,2-b]indole derivatives, all of which were tested in vitro on human CK2. The indenoindolones 5a and 5b inhibited human CK2 with an IC50 of 0.17 and 0.61 µM, respectively. The indeno[1,2-b]indoloquinone 7a also showed inhibitory activity on CK2 at a submicromolar range (IC50 = 0.43 µM). Additionally, a large number of indenoindole derivatives was evaluated for their cytotoxic activities against the cell lines 3T3, WI-38, HEK293T and MEF.