De novo variants of CSNK2B cause a new intellectual disability-craniodigital syndrome by disrupting the canonical Wnt signaling pathway.

CSNK2B encodes for casein kinase II subunit beta (CK2ß), the regulatory subunit of casein kinase II (CK2), which is known to mediate diverse cellular pathways. Variants in this gene have been recently identified as a cause of Poirier-Bienvenu neurodevelopmental syndrome (POBINDS), but functional evidence is sparse. Here, we report five unrelated individuals: two of them manifesting POBINDS, while three are identified to segregate a new intellectual disability-craniodigital syndrome (IDCS), distinct from POBINDS. The three IDCS individuals carried two different de novo missense variants affecting the same codon of CSNK2B. Both variants, NP_001311.3; p.Asp32His and NP_001311.3; p.Asp32Asn, lead to an upregulation of CSNK2B expression at transcript and protein level, along with global dysregulation of canonical Wnt signaling. We found impaired interaction of the two key players DVL3 and ß-catenin with mutated CK2ß. The variants compromise the kinase activity of CK2 as evident by a marked reduction of phosphorylated ß-catenin and consequent absence of active ß-catenin inside nuclei of the patient-derived lymphoblastoid cell lines (LCLs). In line with these findings, whole-transcriptome profiling of patient-derived LCLs harboring the NP_001311.3; p.Asp32His variant confirmed a marked difference in expression of genes involved in the Wnt signaling pathway. In addition, whole-phosphoproteome analysis of the LCLs of the same subject showed absence of phosphorylation for 313 putative CK2 substrates, enriched in the regulation of nuclear ß-catenin and transcription of the target genes. Our findings suggest that discrete variants in CSNK2B cause dominant-negative perturbation of the canonical Wnt signaling pathway, leading to a new craniodigital syndrome distinguishable from POBINDS.

Synthesis and SAR of Tetracyclic Inhibitors of Protein Kinase CK2 Derived from Furocarbazole W16.

The serine/threonine kinase CK2 modulates the activity of more than 300 proteins and thus plays a crucial role in various physiological and pathophysiological processes including neurodegenerative disorders of the central nervous system and cancer. The enzymatic activity of CK2 is controlled by the equilibrium between the heterotetrameric holoenzyme CK2α2 ß2 and its monomeric subunits CK2α and CK2ß. A series of analogues of W16 ((3aR,4S,10S,10aS)-4-{[(S)-4-benzyl-2-oxo-1,3-oxazolidin-3-yl]carbonyl}-10-(3,4,5-trimethoxyphenyl)-4,5,10,10a-tetrahydrofuro[3,4-b]carbazole-1,3(3aH)-dione ((+)-3 a)) was prepared in an one-pot, three-component Levy reaction. The stereochemistry of the tetracyclic compounds was analyzed. Additionally, the chemically labile anhydride structure of the furocarbazoles 3 was replaced by a more stable imide (9) and N-methylimide (10) substructure. The enantiomer (-)-3 a (Ki =4.9 µM) of the lead compound (+)-3 a (Ki =31 µM) showed a more than sixfold increased inhibition of the CK2α/CK2ß interaction (protein-protein interaction inhibition, PPII) in a microscale thermophoresis (MST) assay. However, (-)-3 a did not show an increased enzyme inhibition of the CK2α2 ß2 holoenzyme, the CK2α subunit or the mutated CK2α' C336S subunit in the capillary electrophoresis assay. In the pyrrolocarbazole series, the imide (-)-9 a (Ki =3.6 µM) and the N-methylimide (+)-10 a (Ki =2.8 µM) represent the most promising inhibitors of the CK2α/CK2ß interaction. However, neither compound could inhibit enzymatic activity. Unexpectedly, the racemic tetracyclic pyrrolocarbazole (±)-12, with a carboxy moiety in the 4-position, displays the highest CK2α/CK2ß interaction inhibition (Ki =1.8 µM) of this series of compounds.

Unexpected CK2ß-antagonistic functionality of bisubstrate inhibitors targeting protein kinase CK2.

Protein kinase CK2, a heterotetrameric holoenzyme composed of two catalytic chains (CK2α) attached to a homodimer of regulatory subunits (CK2ß), is a target for drug development for cancer therapy. Here, we describe the tetraiodobenzimidazole derivative ARC-3140, a bisubstrate inhibitor addressing the ATP site and the substrate-binding site of CK2 with extraordinary affinity (Ki = 84 pM). In a crystal structure of ARC-3140 in complex with CK2α, three copies of the inhibitor are visible, one of them at the CK2ß interface of CK2α. Subsequent interaction studies based on microscale thermophoresis and fluorescence anisotropy changes revealed a significant impact of ARC-3140 and of its tetrabromo equivalent ARC-1502 on the CK2α/CK2ß interaction. A structural inspection revealed that ARC-3140, unlike CK2ß antagonists described so far, interferes with both sub-interfaces of the bipartite CK2α/CK2ß interaction. Thus, ARC-3140 is a lead for the further development of highly effective compounds perturbating the quaternary structure of the CK2α2ß2 holoenzyme.

Bacterial Cell-Surface Display of Semisynthetic Cyclic Peptides.

Semisynthetic cyclic peptides containing both non-proteinogenic building blocks, as the synthetic part, and a genetically encoded sequence amenable to DNA-based randomization hold great potential to expand the chemical space in the quest for novel bioactive peptides. Key to an efficient selection of novel binders to biomacromolecules is a robust method to link their genotype and phenotype. A novel bacterial cell surface display technology has been developed to present cyclic peptides composed of synthetic and genetically encoded fragments in their backbones. The fragments were combined by protein trans-splicing and intramolecular oxime ligation. To this end, a split intein half and an unnatural amino acid were displayed with the genetically encoded part on the surface of Escherichia coli. Addition of the synthetic fragment equipped with the split intein partner and an aminooxy moiety, as well as the application of a pH-shift protocol, resulted in the onsurface formation of the semisynthetic cyclic peptide. This approach will serve for the generation of cyclic peptide libraries suitable for selection by fluorescence-activated cell sorting, and more generally enables chemical modification of proteins on the bacterial surface.

Human αS1-casein induces IL-8 secretion by binding to the ecto-domain of the TLR4/MD2 receptor complex.

BACKGROUND: The milk protein αS1-casein was recently reported to induce secretion of proinflammatory cytokines via Toll-like receptor 4 (TLR4). In this study, αS1-casein was identified as binder of theTLR4 ecto domain. METHODS: IL-8 secretion after stimulation of TLR4/MD2 (myeloid differentiation factor 2)/CD14 (cluster of differentiation 14)-transfected HEK293 cells (TLR4+) and Mono Mac 6 cells (MM6) with recombinant αS1-casein, or LPS as control was monitored. Binding of αS1-casein to TLR4 was quantified by microscale thermophoresis (MST). RESULTS: αS1-casein induced secretion of IL-8 in TLR4+ cells and in MM6 cells with a six-times higher final IL-8 concentration in supernatants. IL-8 secretion was inhibited by intracellular TLR4-domain antagonist TAK-242 with an IC50-value of 259.6 nM, by ecto-domain TLR4 antagonistic mianserin with 10-51 µM and by anti-CD14-IgA. The binding constants (KD) of αS1-casein to the TLR4, MD2, and CD14 were 2.8 µM, 0.3 µM and 2.7 µM, respectively. Finally, αS1-casein showed a higher affinity to TLR4/MD2 (KD: 2.2 µM) compared to LPS (KD: 8.2 µM). CONCLUSION: Human αS1-casein induced proinflammatory effects are dependent upon binding to the TLR4 ectodomain and the presence of CD14. αS1-casein displayed stronger TLR4 agonistic activity than LPS via a different mode of action. GENERAL SIGNIFICANCE: Breast milk protein αS1-casein is a proinflammatory cytokine.

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).

Site-Specific Labeling of Protein Kinase CK2: Combining Surface Display and Click Chemistry for Drug Discovery Applications.

Human CK2 is a heterotetrameric constitutively active serine/threonine protein kinase and is an emerging target in current anti-cancer drug discovery. The kinase is composed of two catalytic CK2α subunits and two regulatory CK2ß subunits. In order to establish an assay to identify protein-protein-interaction inhibitors (PPI) of the CK2α/CK2ß interface, a bioorthogonal click reaction was used to modify the protein kinase α-subunit with a fluorophore. By expanding the genetic code, the unnatural amino acid para azidophenylalanine (pAzF) could be incorporated into CK2α. Performing the SPAAC click reaction (Strain-Promoted Azide-Alkyne Cycloaddition) by the use of a dibenzylcyclooctyne-fluorophore (DBCO-fluorophore) led to a specifically labeled human protein kinase CK2α. This site-specific labeling does not impair the phosphorylation activity of CK2, which was evaluated by capillary electrophoresis. Furthermore a dissociation constant (KD) of 631 ± 86.2 nM was determined for the substrate αS1-casein towards CK2α. This labeling strategy was also applied to CK2ß subunit on Escherichia coli, indicating the site-specific modifications of proteins on the bacterial cell surface when displayed by Autodisplay.

Toward selective CK2alpha and CK2alpha' inhibitors: Development of a novel whole-cell kinase assay by Autodisplay of catalytic CK2alpha'.

Human protein kinase CK2 is an emerging target for the development of novel anti-cancer therapeutics. CK2 is a tetramer composed of two catalytically active α- and/or α'-subunits, bound to a dimer of the regulatory ß-subunit. Inhibitors targeting one of the two isoforms of the catalytically active CK2-subunit (α- and α') are important to study the distinct functions of these isoforms toward different CK2 associated pathologies. The present study for the first time describes the successful Autodisplay of the CK2α'-subunit, the paralogous isoform of CK2α. Expression on the cell surface of E. coli of CK2α' alone and in combination with the regulatory CK2ß-subunit was confirmed by outer membrane isolation and protease accessibility test. Kinase activity of surface displayed CK2 could be detected with a CE-based assay and was found to be 3.06×10(-6) µmol/min for CK2α' alone and 1.02×10(-5) µmol/min when expressed in combination with CK2ß. The comparison of the influence of NaCl on activity of the α'-subunit alone and in combination with the non-catalytically active ß-subunit indicated interaction of both subunits on the cell surface. TMCB (4,5,6,7-tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazol-1-yl)acetic acid), a known CK2 inhibitor described with distinct Ki values of 83 nM and 21 nM for the two different catalytic CK2 subunits α and α' was used for testing. First, inhibition of TMCB toward the purified CK2 holoenzyme CK2α2ß2 was determined and resulted in a Ki value of 10.1 nM. Second, Ki values were determined with the surface displayed isoform CK2 holoenzymes and turned out to be of 31.1 nM for CK2α2ß2 and 19.6 nM for CK2α'2ß2. The inhibition data as obtained represented the distinct affinities of TMCB toward the two isoform holoenzymes. This indicated, that the surface display of CKα and CK2α', in the context of the corresponding holoenzymes, can be used to identify selective compounds. A set of twelve ATP competitive CK2 inhibitors with an indeno[1,2-b]indole scaffold was tested in order to demonstrate suitability for this application.

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.