Functional display of heterotetrameric human protein kinase CK2 on Escherichia coli: a novel tool for drug discovery.

BACKGROUND: Human protein kinase CK2 represents a novel therapeutic target for neoplastic diseases. Inhibitors are in need to explore the druggability and the therapeutic options of this enzyme. A bottleneck in the search for new inhibitors is the availability of the target for testing. Therefore an assay was developed to provide easy access to CK2 for discovery of novel inhibitors. RESULTS: Autodisplay was used to present human CK2 on the surface of Escherichia coli. Heterotetrameric CK2 consists of two subunits, α and ß, which were displayed individually on the surface. Co-display of CK2α and CK2ß on the cell surface led to the formation of functional holoenzyme, as demonstrated by NaCl dependency of enzymatic activity, which differs from that of the catalytic subunit CK2α without ß. In addition interaction of CK2α and CK2ß at the cell surface was confirmed by co-immunoprecipitation assays. Surface displayed CK2 holoenzyme enabled an easy IC50 value determination. The IC50 values for the known CK2 inhibitors TBB and Silmitasertib were determined to be 50 and 3.3 nM, respectively. CONCLUSION: Surface-displayed CK2α and CK2ß assembled on the cell surface of E. coli to an active tetrameric holoenzyme. The whole-cell CK2 autodisplay assay as developed is suitable for inhibition studies. Furthermore, it can be used to determine quantitative CK2 inhibition data such as IC50 values. In summary, this is the first report on the functional surface display of a heterotetrameric enzyme on E. coli.

Biologically active carbazole derivatives: focus on oxazinocarbazoles and related compounds.

Four series of carbazole derivatives, including N-substituted-hydroxycarbazoles, oxazinocarbazoles, isoxazolocarbazolequinones, and pyridocarbazolequinones, were studied using diverse biological test methods such as a CE-based assay for CK2 activity measurement, a cytotoxicity assay with IPC-81 cell line, determination of MIC of carbazole derivatives as antibacterial agents, a Plasmodium falciparum susceptibility assay, and an ABCG2-mediated mitoxantrone assay. Two oxazinocarbazoles Ib and Ig showed CK2 inhibition with IC50 = 8.7 and 14.0 µM, respectively. Further chemical syntheses were realized and the 7-isopropyl oxazinocarbazole derivative 2 displayed a stronger activity against CK2 (IC50 = 1.40 µM). Oxazinocarbazoles Ib, Ig, and 2 were then tested against IPC-81 leukemia cells and showed the ability to induce leukemia cell death with IC50 values between 57 and 62 µM. Further investigations were also reported on antibacterial and antiplasmodial activities. No significant inhibitory activity on ABCG2 efflux pump was detected.

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

BACKGROUND: Abnormally 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. METHODS: We 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. RESULTS: The 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 (29 nM) and Ki (15 nM) values. TF inhibited only seven out of 61 human kinases tested (>70% inhibition). Incubation of LNCaP cells with 50 µM TF for 48 h 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 SIGNIFICANCE: In 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.

N-substituted Indole-2 and 3-carboxamide derivatives as inhibitors of human protein kinase CK2: in vitro assay and molecular modelling study.

Protein kinase CK2 (Casein Kinase 2) is involved in cell growth; proliferation and suppression of apoptosis. Hence, it strongly promotes cell survival and can be considered an important target for human cancers. In the present study, a series of N-substituted indole-2- and 3-carboxamide derivatives were tested for inhibitions of human recombinant protein kinase CK2 to evaluate their anticancer properties. The inhibition test revealed that the most active compound 4 (1-benzyl-N-(2,4-dichlorobenzyl)-1H-indole-2-carboxamide) showed an IC50 value of 14.6 µM towards human protein kinase CK2. A molecular docking study of the compounds with CK2 was performed and revealed the binding mode of the most active compound 4, underlying its inhibitory activity.

Identification of novel CK2 inhibitors with a benzofuran scaffold by novel non-radiometric in vitro assays.

Protein kinase CK2 is emerging as a target in neoplastic diseases. Inhibition of CK2 by small compounds could lead to new therapies by counteracting the elevated CK2 activities found in a variety of tumors. Currently, CK2 inhibitors are primarily evaluated by a radiometric in vitro assay tracing the amount of transferred γ-(32)P from ATP to a substrate peptide. Here, we present two alternative assays abandoning radioisotopes. The first assay is based on Förster resonance energy transfer between the fluorescence donor EDANS and the acceptor molecule DABCYL within the CK2 substrate peptide [DABCYL]-RRRDDDSDDD-[EDANS]. This peptide comprises a cleavage site for pancreatic elastase, which is located next to the phosphate acceptor serine. Only the non-phosphorylated peptide can be cleaved by elastase, disrupting the FRET effect. Thus fluorescence intensity is inversely correlated with CK2 activity. The second non-radiometric assay deploys the changing of charge that occurs within the peptide substrate RRRDDDSDDD upon phosphorylation by CK2. Substrate and product of a CK2 reaction consequently show a difference in electrophoretic mobility and thus can be separated by capillary electrophoresis. Absorption detection enabled quantification of both peptide species and allowed the determination of IC(50) values. This method facilitated the testing of a small compound library by which benzofuran derivatives were identified as potent CK2 inhibitors with IC(50) values in the submicromolar range.

A CE-based assay for human protein kinase CK2 activity measurement and inhibitor screening.

A new assay for protein kinase CK2 activity determination based on the quantification of a phosphorylated substrate was developed. The common CK2 substrate peptide RRRDDDSDDD, conjugated with the fluorophore 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid at the C-terminus served as the analyte. By means of CZE using 2 mol/L acetic acid as electrolyte and UV detection at 214 nm, the non-phosphorylated and the phosphorylated peptide variants could be resolved within 6 min from a complex assay mixture. By this means, activity of human CK2 could be monitored by a kinetic, as well as an endpoint, method. Inhibition of human recombinant CK2 holoenzyme by 6-methyl-1,3,8-trihydroxyanthraquinone and 4,5,6,7-tetrabromobenzotriazole resulted in IC(50) values of 1.33 and 0.27 microM, respectively, which were similar to those obtained with the standard radiometric assay. These results suggest that the CE/UV strategy described here is a straightforward assay for CK2 inhibitor testing.

A FRET-based microplate assay for human protein kinase CK2, a target in neoplastic disease.

Besides cardiovascular diseases, cancer represents the major cause of death in developed countries. In many different human tumors, increased activity of serine/threonine protein kinase CK2 has been detected, and recent in vivo studies support a direct involvement of CK2 in tumor progression. Therefore, potent compounds to decrease CK2 activity to a non-pathogenic level would be a promising effort toward an antineoplastic therapy. In this study, an alternative to the established radiometric phosphorylation assay for quantification of CK2 activity was developed. For this purpose, the substrate peptide RRRDDDSDDD was coupled at the C-terminus to the fluorophore EDANS (5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid) and at the N-terminus to the quencher DABCYL (4-(4-dimethylaminophenylazo)benzoic acid). This resulted in quenched fluorescence of EDANS due to a FRET-based effect. After proteolytic cleavage of the peptide by elastase, the quenching effect was reduced and, as a consequence, fluorescence was increased. Because elastase is supposed to cleave at the S/D site of the peptide, phosphorylation of serine by CK2 hampered substrate binding of elastase and blocked the increase in fluorescence by proteolytic cleavage. This means that the new assay to quantify human CK2 activity is based on the differential accessibility of the proteolytic cleavage site, which is dependent on kinase phosphorylation. It could be used to measure inhibition of the human target in neoplastic diseases by the compounds TBB (4,5,6,7-tetrabromobenzotriazole) and Emodin.

Protein domain library generation by overlap extension (PDLGO): a tool for enzyme engineering.

We introduce an upgraded version of the error-prone polymerase chain reaction (epPCR) comprising three DNA polymerase-catalyzed steps. It improves the common epPCR strategy such that random mutations can be confined exactly to a distinct, but freely selectable, sequence region within a gene without the need for flanking restriction endonuclease sites. The new method is called protein domain library generation by overlap extension (PDLGO). To validate PDLGO, we generated a random library of EstE, a multidomain esterase from Xanthomonas vesicatoria. It was demonstrated that random mutations appear exclusively within the catalytic domains as intended. The domains of EstE flanking the catalytic domains are required for transport of EstE to the cell envelope and remain unaltered. Microplates with integrated pH sensors, providing a substrate-independent high-throughput screening tool, were used to analyze whole cells of E. coli expressing the variants of the EstE library. A variant (P286H) with substantially increased catalytic activity was identified. Our results indicate that combining PDLGO with microplates containing integrated pH sensors provides a simple and rapid toolbox for directed evolution of esterases.

A π-Halogen Bond of Dibenzofuranones with the Gatekeeper Phe113 in Human Protein Kinase CK2 Leads to Potent Tight Binding Inhibitors.

Human protein kinase CK2 is an emerging target for neoplastic diseases. Potent lead structures for human CK2 inhibitors are derived from dibenzofuranones. Two new derivatives, 7,9-dichloro-1,2-dihydro-8-hydroxy-4-[(4-methoxyphenylamino)-methylene]dibenzo[b,d]furan-3(2H)-one (4a) and (E)-1,3-dichloro-6-[(4-methoxyphenylimino)-methyl]dibenzo[b,d]furan-2,7-diol (5) were tested for inhibition of CK2 and induction of apoptosis in LNCaP cells. Both turned out to be tight binding inhibitors, with IC50 values of 7 nM (4a) and 5 nM (5) and an apparent Ki value of 0.4 nM for both. Compounds 4a and 5 reduced cellular CK2 activity, indicating cell permeability. Cell viability was substantially impaired in LNCaP cells, as well as apoptosis was induced, which was not appearing in non-neoplastic ARPE-19 cells. Co-crystallization of 4a and 5 revealed an unexpected π-halogen bond of the chloro substituent at C9 with the gatekeeper amino acid Phe113, leading to an inverted binding mode in comparison to parent compound 4b, with the Cl at C6 instead, which was co-crystallized as a control. This indicates that the position of the chloro substituent on ring A of the dibenzofuran scaffold is responsible for an inversion of the binding mode that enhances potency.

Identification of a Potent Allosteric Inhibitor of Human Protein Kinase CK2 by Bacterial Surface Display Library Screening.

Human protein kinase CK2 has emerged as promising target for the treatment of neoplastic diseases. The vast majority of kinase inhibitors known today target the ATP binding site, which is highly conserved among kinases and hence leads to limited selectivity. In order to identify non-ATP competitive inhibitors, a 12-mer peptide library of 6 × 105 variants was displayed on the surface of E. coli by autodisplay. Screening of this peptide library on variants with affinity to CK2 was performed by fluorophore-conjugated CK2 and subsequent flow cytometry. Single cell sorting of CK2-bound E. coli yielded new peptide variants, which were tested on inhibition of CK2 by a CE-based assay. Peptide B2 (DCRGLIVMIKLH) was the most potent inhibitor of both, CK2 holoenzyme and the catalytic CK2α subunit (IC50 = 0.8 µM). Using different ATP concentrations and different substrate concentrations for IC50 determination, B2 was shown to be neither ATP- nor substrate competitive. By microscale thermophoresis (MST) the KD value of B2 with CK2α was determined to be 2.16 µM, whereas no binding of B2 to CK2ß-subunit was detectable. To our surprise, besides inhibition of enzymatic activity, B2 also disturbed the interaction of CK2α with CK2ß at higher concentrations (≥25 µM).

Focusing mutations within random libraries to distinct areas: protein domain library generation by overlap extension.

Directed evolution is an often used approach toward new proteins with tailor-made properties. It consists of random variation of the coding sequence of a protein followed by an appropriate selection procedure or a suitable type of property read out. In many, if not all cases, it is of significant advantage to constrain the randomly mutagenized DNA sequence to that encoding a particular part of the protein or a distinct domain, and not to mutate the entire gene of the target protein. For this purpose, a three-step, polymerase-based method was developed, which is independent of two flanking restriction sites adjacent to the nucleotide sequence supposed to be mutagenized, and named protein library generation by overlap extension (PDLGO).