Inhibitors Of Protein Kinase CK2 Research Articles

Isomeric Mono-, Di-, and Tri-Bromobenzo-1H-Triazoles as Inhibitors of Human Protein Kinase CK2α

To further clarify the role of the individual bromine atoms of 4,5,6,7-tetrabromotriazole (TBBt), a relatively selective inhibitor of protein kinase CK2, we have examined the inhibition (IC50) of human CK2α by the two mono-, the four di-, and the two tri- bromobenzotriazoles relative to that of TBBt. Halogenation of the central vicinal C(5)/C(6) atoms proved to be a key factor in enhancing inhibitory activity, in that 5,6-di-Br2Bt and 4,5,6-Br3Bt were almost as effective inhibitors as TBBt, notwithstanding their marked differences in pKa for dissociation of the triazole proton. The decrease in pKa on halogenation of the peripheral C(4)/C(7) atoms virtually nullifies the gain due to hydrophobic interactions, and does not lead to a decrease in IC50. Molecular modeling of structures of complexes of the ligands with the enzyme, as well as QSAR analysis, pointed to a balance of hydrophobic and electrostatic interactions as a discriminator of inhibitory activity. The role of halogen bonding remains debatable, as originally noted for the crystal structure of TBBt with CK2α (pdb1j91). Finally we direct attention to the promising applicability of our series of well-defined halogenated benzotriazoles to studies on inhibition of kinases other than CK2.

4,5,6,7-Tetrachloro-2-(1H-imidazol-2-yl)isoindoline-1,3-dione

4,5,6,7-Tetrachloro-2-(1H-imidazol-2-yl)isoindoline-1,3-dione was obtained by reaction of 2-aminoimidazole with 3,4,5,6-tetrachlorophthalic anhydride in refluxing acetic acid.

The Role of Protein Kinase CK2 (PKCK2) in the Cyclosporine Induced Nephropathy in Rats

Background: Protein kinase casein kinase II (PKCK2) has multiple, overlapping roles in induction of apoptosis. Apoptosis might to be a common pathway of renal injury caused by nephrotoxic drug or injury. We evaluated the role of PKCK2 in the Cyclosporin A (CsA) induced nephropathy in rats by inhibition of PKCK2. Methods: Male Sprague Dawley rats fed with low sodium diet were divided into four treatment groups: control (0.9% saline injection), CsA (15mg/kg/day by s.c.), CsA + emodin (CsA plus emodin 20mg/kg/day by s.c.), and emodin only. The expression levels of apoptosis-associated factors were examined by western blot analysis. Expression of PKCK2 was also assayed by western blot. Results: Overexpression of PKCK2 was noted in CsA treatment. Emodin, a low molecular weight PKCK2 specific inhibitor, prevented the up-regulation of phosphorylated p53 and the activation of caspase 3, 7, and 8 induced by CsA treatment. In addition, emodin prevented CsA-induced increased expression of Bax/Bcl-2 ratio. Emodin prevented the up-regulation of PKCK2 induced by CsA treatment, indicating that its apoptotic-preventing activity was mediated via PKCK2. Conclusions: Our findings indicate that PKCK2 may play a major role in apoptotic injury in CsA induced nephropathy in rat models.

Functional proteomics strategy for validation of protein kinase inhibitors reveals new targets for a TBB-derived inhibitor of protein kinase CK2

CK2 is a constitutively active protein kinase with key regulatory roles in many cellular signaling events which has been implicated in several human diseases. To investigate its roles in biological events and potential as a therapeutic target, several potent CK2 inhibitors have been developed including TBB and its derivatives that have been employed in many studies. Despite the utility of these compounds, a precise understanding of their mode of action within cells remains incomplete. In fact, cells are typically treated with inhibitor concentrations (>5 μM) that are orders of magnitude higher than their in vitro inhibitory constants (<0.05 μM). Accordingly, we hypothesized that CK2 inhibitors could have off-target effects that are not recognized when inhibitors are profiled using panels of recombinant protein kinases. To address this issue, we combined structural modeling with inhibitor-affinity purification and proteomics to test the specificity of derivatives of TBB using whole cell lysates of HeLa cells. While these studies confirmed that CK2 does bind to the immobilized inhibitor, several other abundant ATP/GTP-binding proteins were also identified and confirmed. These results suggest that highly abundant nucleotide binding proteins may limit the bioavailability of the free inhibitor and interactions with CK2 in the cellular environment. This article is part of a Special Issue entitled: From protein structures to clinical applications.

Inhibition of Protein Kinase CK2 by Flavonoids and Tyrphostins. A Structural Insight

Sixteen flavonoids and related compounds have been tested for their ability to inhibit three acidophilic Ser/Thr protein kinases: the Golgi apparatus casein kinase (G-CK) recently identified with protein FAM20C, protein kinase CK1, and protein kinase CK2. While G-CK is entirely insensitive to all compounds up to 40 μM concentration, consistent with the view that it is not a member of the kinome, and CK1 is variably inhibited in an isoform-dependent manner by fisetin and luteolin, and to a lesser extent by myricetin and quercetin, CK2 is susceptible to drastic inhibition by many flavonoids, displaying with six of them IC(50) values < 1 μM. A common denominator of these compounds (myricetin, quercetin, fisetin, kaempferol, luteolin, and apigenin) is a flavone scaffold with at least two hydroxyl groups at positions 7 and 4'. Inhibition is competitive with respect to the phospho-donor substrate ATP. The crystal structure of apigenin and luteolin in complex with the catalytic subunit of Zea mays CK2 has been solved, revealing their ability to interact with both the hinge region (Val116) and the positive area near Lys68 and the conserved water W1, the two main polar ligand anchoring points in the CK2 active site. Modeling experiments account for the observation that luteolin but not apigenin inhibits also CK1. The observation that luteolin shares its pyrocatechol moiety with tyrphostin AG99 prompted us to solve also the structure of this compound in complex with CK2. AG99 was found inside the ATP pocket, consistent with its mode of inhibition competitive with respect to ATP. As in the case of luteolin, the pyrocatechol group of AG99 is critical for binding, interacting with the positive area in the deepest part of the CK2 active site.

Novel indeno[1,2-b]indoloquinones as inhibitors of the human protein kinase CK2 with antiproliferative activity towards a broad panel of cancer cell lines

We previously reported indeno[1,2-b]indoles as a novel class of potent inhibitors of the human protein kinase CK2. In the present study we prepared two novel quinoid derivatives, the indeno[1,2-b]indoloquinones 6b and 6c, and demonstrated inhibition of the human CK2 by the compounds. Furthermore, we showed substantial antiproliferative activity of both compounds towards a broad panel of human cancer cell lines in the low micromolar range. Whereas the earlier indeno[1,2-b]indoles have been shown to be selective for CK2, the indeno[1,2-b]indoloquinones 6b and 6c also inhibited the AMPK activated protein kinase ARK5, potentially contributing to the anti-cancer effects of the compounds. In addition, with compound 6b we found a very potent inhibitor of the leukemia-associated receptor tyrosine kinase FLT3, with an IC50 of 0.18μM.

Discovery and structure–activity relationship of 2,6-disubstituted pyrazines, potent and selective inhibitors of protein kinase CK2

We report the discovery and structure–activity relationship of 2,6-disubstituted pyrazines, which are potent and selective CK2 inhibitors. Lead compound 1 was identified, and derivatives were prepared to develop potent inhibitory activity. As a result, we obtained compound 7, which was the smallest unit that retained potency. Then, introducing an aminoalkyl group at the 6-position of the indazole ring resulted in improved efficacy in both enzymatic and cell-based CK2 inhibition assays. Moreover, compound 13 showed selectivity against other kinases and in vivo efficacy in a rat nephritis model. These results show that 2,6-disubstituted pyrazines have potential as therapeutic agents for nephritis.

Casein Kinase 2-mediated Synaptic GluN2A Up-regulation Increases N-Methyl-d-aspartate Receptor Activity and Excitability of Hypothalamic Neurons in Hypertension

Increased glutamatergic input, particularly N-methyl-D-aspartate receptor (NMDAR) activity, in the paraventricular nucleus (PVN) of the hypothalamus is closely associated with high sympathetic outflow in essential hypertension. The molecular mechanisms underlying augmented NMDAR activity in hypertension are unclear. GluN2 subunit composition at the synaptic site critically determines NMDAR functional properties. Here, we found that evoked NMDAR-excitatory postsynaptic currents (EPSCs) of retrogradely labeled spinally projecting PVN neurons displayed a larger amplitude and shorter decay time in spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto (WKY) rats. Blocking GluN2B caused a smaller decrease in NMDAR-EPSCs of PVN neurons in SHRs than in WKY rats. In contrast, GluN2A blockade resulted in a larger reduction in evoked NMDAR-EPSCs and puff NMDA-elicited currents of PVN neurons in SHRs than in WKY rats. Blocking presynaptic GluN2A, but not GluN2B, significantly reduced the frequency of miniature EPSCs and the firing activity of PVN neurons in SHRs. The mRNA and total protein levels of GluN2A and GluN2B in the PVN were greater in SHRs than in WKY rats. Furthermore, the GluN2B Ser(1480) phosphorylation level and the synaptosomal GluN2A protein level in the PVN were significantly higher in SHRs than in WKY rats. Inhibition of protein kinase CK2 normalized the GluN2B Ser(1480) phosphorylation level and the contribution of GluN2A to NMDAR-EPSCs and miniature EPSCs of PVN neurons in SHRs. Collectively, our findings suggest that CK2-mediated GluN2B phosphorylation contributes to increased synaptic GluN2A, which potentiates pre- and postsynaptic NMDAR activity and the excitability of PVN presympathetic neurons in hypertension.

Abstract 3765: Discovery and biological characterization of CX-8184, a potent inhibitor of protein kinase CK2

Abstract Protein kinase CK2 regulates multiple pathways that play key roles in carcinogenesis, including cell cycle progression, suppression of apoptosis, hypoxia, angiogenesis, inflammation and DNA damage repair. Overexpression of CK2 in various cancer tissues has been frequently linked to poor prognosis. All this makes targeting of CK2 highly attractive for both single agent and combination therapies. To date therapeutic targeting of CK2 proved to be challenging, with only one compound, CX-4945, entering clinical development. In phase I clinical trials CX-4945 was well tolerated, inhibited CK2 activity in PBMC, modulated the PI3K-AKT-mTOR oncogenic pathway, reduced tumor-related serum IL-6 and IL-8 biomarkers, resulted in stable disease for at least 16 weeks in 20% of treated patients, and caused single agent tumor shrinkage. Building on these successes with the first generation CK2 inhibitor, we sought to design a second generation CK2 inhibitor with distinct pharmacologic properties. The application of a focused screening cascade and iterative medicinal chemistry efforts generated CX-8184, a selective single digit nanomolar inhibitor of the CK2 enzyme that demonstrated desirable biologic properties. When evaluated against a panel of 32 cancer cell lines, CX-8184 demonstrated broad antiproliferative activity with median IC50 = 145 nM. CX-8184 suppressed NF-κB, Hif-1α and β-catenin driven transcription at concentrations of 100-300 nM, and inhibited IL-6 expression with IC50 = 30 nM. Since CK2 is known to potentiate AKT signaling, treatment of cancer cells with CX-8184 decreased phosphorylation of AKT, as well as its substrates. The Hsp90/Cdc37 machinery is regulated by CK2 and plays an important role in activation of various kinases, including members of the ErbB RTK family. CX-8184 rapidly suppressed phosphorylation of Cdc37 by CK2, leading to the deactivation of ErbB family members. In combination with erlotinib or lapatinib, CX-8184 produced synergistic killing of lung cancer and breast cancer cells, respectively. CX-8184 also inhibited the phosphorylation of XRCC1, a scaffold protein involved in DNA strand break repair that plays an important role in sensitivity of cancer cells to DNA damaging agents. Consequently, the treatment of ovarian cancer cells pre-challenged with gemcitabine and/or cisplatin with 300 nM CX-8184 led to synergistic induction of cell death. In vivo, CX-8184 demonstrated favorable PK properties with oral bioavailability and a half-life amenable to once daily dosing in mice. CX-8184 exhibited potent anti-tumor activity and was well tolerated in A431 xenograft model. The compound has also showed an acceptable pharmacological profile as evidenced by CYP, hERG and Ames testing. The pharmacokinetic and pharmacological properties and the biological activity of CX-8184, both as single agent or in combination, support its further development as a preclinical candidate.9 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3765. doi:1538-7445.AM2012-3765

Structure-Based Design of Novel Potent Protein Kinase CK2 (CK2) Inhibitors with Phenyl-azole Scaffolds

Protein kinase CK2 (CK2) is a ubiquitous serine/threonine protein kinase for hundreds of endogenous substrates. CK2 has been considered to be involved in many diseases, including cancers. Herein we report the discovery of a novel ATP-competitive CK2 inhibitor. Virtual screening of a compound library led to the identification of a hit 2-phenyl-1,3,4-thiadiazole compound. Subsequent structural optimization resulted in the identification of a promising 4-(thiazol-5-yl)benzoic acid derivative.

TF — A novel cell-permeable and selective inhibitor of human protein kinase CK2 induces apoptosis in the prostate cancer cell line LNCaP

BackgroundAbnormally high activity of protein kinase CK2 is linked to various diseases including cancer. Therefore, the inhibition of CK2 is a promising therapeutic strategy to fight this disease. MethodsWe screened a library of synthetic molecules concerning their capacity to inhibit CK2. The activity of CK2 and their IC50 and Ki values were determined by a capillary electrophoresis assay. The effects of the inhibitor in a cell culture model were analyzed by cell counting, a viability assay, cytofluorimetry and Western blot. ResultsThe best CK2 inhibitor found in this screen was 6,7-dichloro-1,4-dihydro-8-hydroxy-4-[(4-methylphenylamino)methylen]dibenzo [b,d]furan-3(2H)-one, which we refer to as “TF”. TF showed tight binding to CK2 with low IC50 (29nM) and Ki (15nM) values. TF inhibited only seven out of 61 human kinases tested (>70% inhibition). Incubation of LNCaP cells with 50μM TF for 48h decreased the intracellular CK2 activity by 50%, confirming that the inhibitor is membrane permeable. The decrease in activity was correlated with a severe reduction in cell viability. The reduction in cell viability is at least partly due to the induction of apoptosis. General significanceIn many cancers the protein kinase CK2 is significantly up-regulated and supports the neoplastic phenotype. New therapeutic strategies should be based on diverse reliable inhibitors to reverse the abnormal high levels to normal settings.

Indeno[1,2-b]indole derivatives as a novel class of potent human protein kinase CK2 inhibitors

Herein we describe the synthesis and properties of indeno[1,2-b]indole derivatives as a novel class of potent inhibitors of the human protein kinase CK2. A set of 19 compounds was obtained using a convenient and straightforward synthesis protocol. The compounds were tested for inhibition of human protein kinase CK2, which was recombinantly expressed in Escherichia coli. New inhibitors with IC50 in the micro- and sub-micromolar range were identified. Compound 4b (5-isopropyl-7,8-dihydroindeno[1,2-b]indole-9,10(5H,6H)-dione) inhibited human CK2 with an IC50 of 0.11μM and did not significantly inhibit 22 other human protein kinases, suggesting selectivity towards CK2. ATP-competitive inhibition by compound 4b was shown and a Ki of 0.06μM was determined. Our findings indicate that indeno[1,2-b]indoles are a promising starting point for further development and optimization of human protein kinase CK2 inhibitors.

Comparative analysis of stress responses of H9c2 rat cardiomyoblasts following treatment with doxorubicin and tBOOH

Cardiotoxicity is the major dose-limiting adverse effect of anthracyclines and is hypothesized to result from damage induced by reactive oxygen species (ROS) or inhibition of topoisomerase II. Here, we comparatively analyzed the effect of doxorubicin and the organic peroxide tertiary-butylhydroperoxide (tBOOH) on stress responses of rat cardiomyblast cells (H9c2). Moreover, we investigated the impact of serum factors and the novel prototypical protein kinase CK2 inhibitor resorufin on the sensentivity of H9c2 cells exposed to doxorubicin or tBOOH. Measuring cell viability by use of the WST assay as well as cell cycle progression and apoptotic death by FACS-based methods, we found that the sensitivity of H9c2 cells to doxorubicin and tBOOH was differently affected by both serum factors and resorufin. Formation of reactive oxygen species was observed after exposure of H9c2 cells to high doses (i.e. ≥5μM) of doxorubicin only. Moreover, the antioxidant N-acetylcysteine protected H9c2 cells from cytotoxicity provoked by tBOOH but not doxorubicin. Analyzing the phosphorylation level of genotoxic stress responsive protein kinases and histone H2AX, which is indicative of an activated DNA damage response (DDR), we found that resorufin modulates doxorubicin- and tBOOH-induced responses in an agent specific manner. Taken together, the data indicate that (i) oxidative injury is not the most relevant type of damage triggering cell death of H9c2 cells following doxorubicin treatment, (ii) serum factors differently influence the sensitivity of cardiomyoblasts to doxorubicin and tBOOH and (iii) inhibition of CK2 unequally affects doxorubicin- and tBOOH-induced DDR of rat cardiomyoblasts.

Antitumoral activity of allosteric inhibitors of protein kinase CK2.

Due to its physiological role into promoting cell survival and its dysregulation in most cancer cells, protein kinase CK2 is a relevant physiopathological target for development of chemical inhibitors. We report the discovery of azonaphthalene derivatives, as a new family of highly specific CK2 inhibitors. First, we demonstrated that CK2 inhibition (IC50= 0.4 µM) was highly specific, reversible and non ATP-competitive. Small Angle X-ray Scattering experiments showed that this inhibition was due to large conformational change of CK2α upon binding of these inhibitors. We showed that several compounds of the family were cell-potent CK2 inhibitors promoting cell cycle arrest of human glioblastoma U373 cells. Finally, in vitro and in vivo assays showed that these compounds could decrease U373 cell tumor mass by 83 % emphasizing their efficacy against these apoptosis-resistant tumors. In contrast, Azonaphthalene derivatives inactive on CK2 activity showed no effect in colony formation and tumor regression assays. These findings illustrate the emergence of nonclassical CK2 inhibitors and provide exciting opportunities for the development of novel allosteric CK2 inhibitors. CK2 is an emerging therapeutic target and ATP-competitive inhibitors have been identified. CK2 is endowed with specific structural features providing alternative strategies for inhibition. Azonaphthalene compounds are allosteric CK2 inhibitors showing antitumor activity. CK2 may be targeted allosterically. These inhibitors provide a foundation for a new paradigm for specific CK2 inhibition.

Cover Picture: Urolithin as a Converging Scaffold Linking Ellagic acid and Coumarin Analogues: Design of Potent Protein Kinase CK2 Inhibitors (ChemMedChem 12/2011)

The cover picture shows the hypothetical binding motif of a novel potent protein kinase CK2 inhibitor, 4-bromo-3,8-dihydroxy-benzo[c]chromen-6-one, which shows a Ki value of 7 nM. CK2 is a ubiquitous, essential, and highly pleiotropic protein kinase, and abnormally high constitutive activity of this enzyme is thought to mediate its pathogenic potential in neoplasia and other diseases, such as viral infections and inflammatory diseases, like glomerulonephritis. Starting from the previous identification of highly active 3,8-dibromo-7-hydroxy-4-methylchromen-2-one and ellagic acid (Ki=20 nM and 60 nM, respectively), an X-ray-driven “cut & paste approach” was applied to design a novel series of urolithins as potent CK2 inhibitors. For more details, see the Full Paper by Stefano Moro et al. on p. 2273 ff.

Synthesis and SAR of inhibitors of protein kinase CK2: Novel tricyclic quinoline analogs

Protein kinase CK2 is a potential drug target for many diseases including cancer and inflammation disorders. The crystal structure of clinical candidate CX-4945 1 with CK2 revealed an indirect interaction with the protein through hydrogen bonding between the NH of the 3-chlorophenyl amine and a water molecule. Herein, we investigate the relevance of this hydrogen bond by preparing several novel tricyclic derivatives lacking a NH moiety at the same position. This SAR study allowed the discovery of highly potent CK2 inhibitors.

Down-Regulation of NF-κB and STAT3 Signaling Pathways Upon Protein Kinase CK2 Inhibition Empowers Bortezomib-Induced Multiple Myeloma Cell-Death

Abstract 1849Multiple Myeloma (MM) malignant plasma cells can be induced to die by blocking the proteasome. Bortezomib (BZ), a first-in class proteasome inhibitor of wide clinical use in MM patients, causes MM cell apoptosis through different mechanisms; however, the means of resistance to its effects are poorly recognized. Our group identified protein kinase CK2 as a critical survival molecule for MM cells (Piazza FA et al., 2006, Blood, 108(5):1698–707). This kinase regulates pivotal apoptosis-related pathways in cancer cells; however, it is currently unknown whether CK2 could be involved downstream proteasome inhibition. Intriguingly, phase I clinical trials are currently ongoing with an oral ATP-competitive CK2 inhibitor in MM and other tumors. We have here sought to investigate whether CK2 takes part in BZ-induced MM cell apoptosis and we studied whether blocking CK2 could influence pro-survival signalling pathways, which could account for MM cell resistance to BZ and chemotherapy.MM cell lines U-266, RPMI-8226 and INA-6, human bone marrow stromal cells and freshly isolated plasma cells from patients were cultured and exposed to BZ and CK2 inhibitors K27 and CX4945 for different time points. Annexin V and propidium iodide staining, evaluation of mitochondrial membrane potential depolarization and western blot (WB) analysis of PARP cleavage and apoptosis-related proteins expression were the assays employed to assess cell growth and viability upon the different treatments.We found that the rate of BZ-induced MM cell apoptosis was significantly increased by the simultaneous inhibition of CK2 and the proteasome in all the MM models tested and mitochondrial membrane potential measurements revealed that CK2 inhibition enhanced BZ-triggered intrinsic apoptotic cascade. Importantly, the combination of CK2 inhibitors and BZ resulted in a synergic growth-suppressive action. WB and RT-PCR analysis revealed that survival-signalling pathways associated with STAT3 and NF-κB were activated by BZ, which also caused a rise in the levels of the unfolded protein response-associated kinase/endoribonuclease IRE1α. These effects could represent unwanted side consequences of BZ treatment and could lend MM cells the ability to escape the cytotoxic effects of this drug. CK2 inhibition produced a strong reduction of phospho Ser 536 and phospho Ser 529 p65 NF-κB subunit, phospho Ser 727 STAT3 and IRE1α levels in MM cells. Remarkably, the simultaneous treatment with BZ and CK2 inhibitors was accompanied by a significant reduction of BZ-triggered p65 NF-κB and STAT3 activation and IRE1α protein levels.These results indicate that protein kinase CK2 protects from BZ-induced apoptosis and modulates pivotal signaling pathways in MM cells, such as the NF-κB and STAT3 cascades, which could otherwise be exploited in the selection of BZ-resistant MM cell clones. Our findings suggest that CK2 inhibition could offer a rational therapeutic option when designing novel BZ-based anti-MM combination therapies. Disclosures:No relevant conflicts of interest to declare.

Structural Determinants of CX-4945 Derivatives as Protein Kinase CK2 Inhibitors: A Computational Study

Protein kinase CK2, also known as casein kinase-2, is involved in a broad range of physiological events including cell growth, proliferation and suppression of apoptosis which are related to human cancers. A series of compounds were identified as CK2 inhibitors and their inhibitory activities varied depending on their structures. In order to explore the structure-activity correlation of CX-4945 derivatives as inhibitors of CK2, in the present study, a set of ligand- and receptor-based 3D-QSAR models were developed employing Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Index Analysis (CoMSIA). The optimum CoMFA (Rcv2 = 0.618, Rpred2 = 0.892) and CoMSIA (Rcv2 = 0.681, Rpred2 = 0.843) models exhibited reasonable statistical characteristics for CX-4945 derivatives. The results indicated that electrostatic effects contributed the most to both CoMFA and CoMSIA models. The combination of docking analysis and molecular dynamics (MD) simulation showed that Leu45, Lys68, Glu81, Val116, Asp175 and Trp176 of CK2 which formed several direct or water-bridged H-bonds with CX-4945 are crucial for CX-4945 derivatives recognition to CK2. These results can offer useful theoretical references for designing more potent CK2 inhibitors.

Unprecedented Selectivity and Structural Determinants of a New Class of Protein Kinase CK2 Inhibitors in Clinical Trials for the Treatment of Cancer

5-(3-Chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic acid (CX-4945), the first clinical stage inhibitor of protein kinase CK2 for the treatment of cancer, is representative of a new class of CK2 inhibitors with K(i) values in the low nanomolar range and unprecedented selectivity versus other kinases. Here we present the crystal structure of the complexes of CX-4945 and two analogues (CX-5011 and CX-5279) with the catalytic subunit of human CK2. Consistent with their ATP-competitive mode of inhibition, all three compounds bind in the active site of CK2 (type I inhibitors). The tricyclic scaffold of the inhibitors superposes on the adenine of ATP, establishing multiple hydrophobic interactions with the binding cavity. The more extended scaffold, as compared to that of ATP, allows the carboxylic function, shared by all three ligands, to penetrate into the deepest part of the active site where it makes interactions with conserved water W1 and Lys-68, thus accounting for the crucial role of this negatively charged group in conferring high potency to this class of inhibitors. The presence of a pyrimidine in CX-5011 and in CX-5279 instead of a pyridine (as in CX-4945) ring is likely to account for the higher specificity of these compounds whose Gini coefficients, calculated by profiling them against panels of 102 and/or 235 kinases, are significantly higher than that of CX-4945 (0.735 and 0.755, respectively, vs 0.615), marking the highest selectivity ever reported for CK2 inhibitors.

Multisite-directed inhibitors of protein kinase CK2: new challenges

New 4,5,6,7-tetrabromo benzotriazole derivatives have been synthesized, and their activities against CK2 have been tested. A click chemistry approach based on the copper-catalyzed azide-alkyne cycloaddition has been utilized to connect benzotriazoles, which efficiently interact with the ATP-binding site, to other subunits designed to simultaneously bind to the active and the substrate-binding sites of the enzyme. Docking studies allowed us to identify key interactions between CK2 and the designed ligands, which will be useful to optimize this series of multisite-directed inhibitors.