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.

Application of an alchemical free energy method for the prediction of thermostable DuraPETase variants.

Non-equilibrium (NEQ) alchemical free energy calculations are an emerging tool for accurately predicting changes in protein folding free energy resulting from amino acid mutations. In this study, this method in combination with the Rosetta ddg monomer tool was applied to predict more thermostable variants of the polyethylene terephthalate (PET) degrading enzyme DuraPETase. The Rosetta ddg monomer tool efficiently enriched promising mutations prior to more accurate prediction by NEQ alchemical free energy calculations. The relative change in folding free energy of 96 single amino acid mutations was calculated by NEQ alchemical free energy calculation. Experimental validation of ten of the highest scoring variants identified two mutations (DuraPETaseS61M and DuraPETaseS223Y) that increased the melting temperature (Tm) of the enzyme by up to 1 °C. The calculated relative change in folding free energy showed an excellent correlation with experimentally determined Tm resulting in a Pearson's correlation coefficient of r = - 0.84. Limitations in the prediction of strongly stabilizing mutations were, however, encountered and are discussed. Despite these challenges, this study demonstrates the practical applicability of NEQ alchemical free energy calculations in prospective enzyme engineering projects. KEY POINTS: • Rosetta ddg monomer enriches stabilizing mutations in a library of DuraPETase variants • NEQ free energy calculations accurately predict changes in Tm of DuraPETase • The DuraPETase variants S223Y, S42M, and S61M have increased Tm.

Structural Analysis of Breast-Milk αS1-Casein: An α-Helical Conformation Is Required for TLR4-Stimulation.

Breast-milk αS1-casein is a Toll-like receptor 4 (TLR4) agonist, whereas phosphorylated αS1-casein does not bind TLR4. The objective of this study was to analyse the structural requirements for these effects. In silico analysis of αS1-casein indicated high α-helical content with coiled-coil characteristics. This was confirmed by CD-spectroscopy, showing the α-helical conformation to be stable between pH 2 and 7.4. After in vitro phosphorylation, the α-helical content was significantly reduced, similar to what it was after incubation at 80 °C. This conformation showed no in vitro induction of IL-8 secretion via TLR4. A synthetic peptide corresponding to V77-E92 of αS1-casein induced an IL-8 secretion of 0.95 ng/mL via TLR4. Our results indicate that αS1-casein appears in two distinct conformations, an α-helical TLR4-agonistic and a less α-helical TLR4 non-agonistic conformation induced by phosphorylation. This is to indicate that the immunomodulatory role of αS1-casein, as described before, could be regulated by conformational changes induced by phosphorylation.

Controlled E. coli Aggregation Mediated by DNA and XNA Hybridization.

Chemical cell surface modification is a fast-growing field of research, due to its enormous potential in tissue engineering, cell-based immunotherapy, and regenerative medicine. However, engineering of bacterial tissues by chemical cell surface modification has been vastly underexplored and the identification of suitable molecular handles is in dire need. We present here, an orthogonal nucleic acid-protein conjugation strategy to promote artificial bacterial aggregation. This system gathers the high selectivity and stability of linkage to a protein Tag expressed at the cell surface and the modularity and reversibility of aggregation due to oligonucleotide hybridization. For the first time, XNA (xeno nucleic acids in the form of 1,5-anhydrohexitol nucleic acids) were immobilized via covalent, SNAP-tag-mediated interactions on cell surfaces to induce bacterial aggregation.

Autodisplay of streptococcal protein G for construction of an orientation-controlled immunoaffinity layer.

An immunoaffinity layer with orientation-controlled antibodies was constructed to express streptococcal protein G in Escherichia coli cells using autodisplay technology. The sequence of protein G, a specific IgG-binding protein, was inserted into the autodisplay vector using recombinant technology and the constructed plasmid vector was transformed into E. coli cells. Protein G was confirmed to be autodisplayed with a high density of 2 × 105 copies per cell by SDS-PAGE analysis, and its IgG-binding affinity was confirmed by fluorescence microscopy. Autodisplayed protein G showed higher affinity than the IgG-binding Z-domain for goat IgG. Immunoassays based on E. coli cells were established to detect horseradish peroxidase (HRP) and C-reactive protein (CRP). Protein G autodisplaying E. coli cells were utilized as a solid support and immunoassays showed improved sensitivity by orientation control of autodisplayed protein G. The outer membrane (OM) of protein G autodisplaying E. coli was isolated and layered to construct an immunoaffinity layer. The OM was coated on a microplate to perform the immunoassays, which showed limits of detection of 5 and 0.2 ng mL-1 for HRP and CRP, respectively. An OM layer with autodisplayed protein G was applied as the immunoaffinity layer of a surface plasmon resonance (SPR) biosensor. After CRP detection, the SPR responses showed good linearity, with an R2 value of 0.99. The immunoaffinity layer with orientation control by autodisplayed protein G was confirmed to be applicable in immunoassays and immunosensors to improve sensitivity.

A FRET-Based Assay for the Identification of PCNA Inhibitors.

Proliferating cell nuclear antigen (PCNA) is the key regulator of human DNA metabolism. One important interaction partner is p15, involved in DNA replication and repair. Targeting the PCNA-p15 interaction is a promising therapeutic strategy against cancer. Here, a Förster resonance energy transfer (FRET)-based assay for the analysis of the PCNA-p15 interaction was developed. Next to the application as screening tool for the identification and characterization of PCNA-p15 interaction inhibitors, the assay is also suitable for the investigation of mutation-induced changes in their affinity. This is particularly useful for analyzing disease associated PCNA or p15 variants at the molecular level. Recently, the PCNA variant C148S has been associated with Ataxia-telangiectasia-like disorder type 2 (ATLD2). ATLD2 is a neurodegenerative disease based on defects in DNA repair due to an impaired PCNA. Incubation time dependent FRET measurements indicated no effect on PCNAC148S-p15 affinity, but on PCNA stability. The impaired stability and increased aggregation behavior of PCNAC148S was confirmed by intrinsic tryptophan fluorescence, differential scanning fluorimetry (DSF) and asymmetrical flow field-flow fractionation (AF4) measurements. The analysis of the disease associated PCNA variant demonstrated the versatility of the interaction assay as developed.

In Silico and In Vitro Search for Dual Inhibitors of the Trypanosoma brucei and Leishmania major Pteridine Reductase 1 and Dihydrofolate Reductase.

The parasites Trypanosoma brucei (Tb) and Leishmania major (Lm) cause the tropical diseases sleeping sickness, nagana, and cutaneous leishmaniasis. Every year, millions of humans, as well as animals, living in tropical to subtropical climates fall victim to these illnesses' health threats. The parasites' frequent drug resistance and widely spread natural reservoirs heavily impede disease prevention and treatment. Due to pteridine auxotrophy, trypanosomatid parasites have developed a peculiar enzyme system consisting of dihydrofolate reductase-thymidylate synthase (DHFR-TS) and pteridine reductase 1 (PTR1) to support cell survival. Extending our previous studies, we conducted a comparative study of the T. brucei (TbDHFR, TbPTR1) and L. major (LmDHFR, LmPTR1) enzymes to identify lead structures with a dual inhibitory effect. A pharmacophore-based in silico screening of three natural product databases (approximately 4880 compounds) was performed to preselect possible inhibitors. Building on the in silico results, the inhibitory potential of promising compounds was verified in vitro against the recombinant DHFR and PTR1 of both parasites using spectrophotometric enzyme assays. Twelve compounds were identified as dual inhibitors against the Tb enzymes (0.2 µM < IC50 < 85.1 µM) and ten against the respective Lm enzymes (0.6 µM < IC50 < 84.5 µM). These highly promising results may represent the starting point for the future development of new leads and drugs utilizing the trypanosomatid pteridine metabolism as a target.

Antibody-Mediated Screening of Peptide Inhibitors for Monoamine Oxidase-B (MAO-B) from an Autodisplayed FV Library.

Inhibitors for monoamine oxidase-B (MAO-B) were screened from an FV library with a randomized complementarity-determining region 3 (CDR3) region using a monoclonal antibody against dopamine. As the first step, the FV library was expressed on the outer membrane of E. coli by site-directed mutagenesis of the randomized CDR3 region. Among the FV library, variants with a binding affinity to monoclonal antibodies against dopamine were screened and cloned. From the comparison of the binding activity of the screened clones to a control clone with a modified FV antibody (only with CDR1 and CDR2), the CDR3 regions of screened clones were determined to directly interact with the monoclonal antibody against dopamine. These CDR3 sequences were then synthesized as mimotopes (mimicking peptides) of dopamine. The inhibitory activity of two mimotopes against MAO-B was analyzed using HeLa cells overexpressing MAO-B, as well as using activated human astrocytes; their inhibitory activity was compared to that of a commercial inhibitor of MAO-B, selegiline. The inhibition efficiency of the two mimotopes (in comparison with selegiline) was estimated to be 67.2% and 69.4% in the HeLa cells and 64.4% and 58.0% in the human astrocytes. The gene expression pattern in astrocytes after treatment with the two mimotopes was also analyzed and compared with that in the human astrocytes treated with selegiline. Finally, the interaction between two mimotopes and MAO-B was analyzed using docking simulation, and the candidate regions of MAO-B for the interaction with each mimotope were explored through the docking simulation.

Fluorescein and Rhodamine B-Binding Domains from Autodisplayed Fv-Antibody Library.

In this study, the binding domains for fluorescent dyes were presented that could be used as synthetic peptides or fusion proteins. Fv-antibodies against two fluorescent dyes (fluorescein and rhodamine B) were screened from the Fv-antibody library, which was prepared on the outer membrane of Escherichia coli using the autodisplay technology. Two clones with binding activities to each fluorescent dye were screened separately from the library using flow cytometry. The binding activity of the screened Fv-antibodies on the outer membrane was analyzed using fluorescent imaging with the corresponding fluorescent dyes. The CDR3 regions of the screened Fv-antibodies (11 amino acid residues) were synthesized into peptides, and each peptide was analyzed for its binding activity to each fluorescent dye using fluorescence resonance energy transfer (FRET) experiments. These CDR3 regions were demonstrated to have a binding activity to each fluorescent dye when the regions were co-expressed as a fusion protein with Z-domain.

Covalently Immobilized Regenerable Immunoaffinity Layer with Orientation-Controlled Antibodies Based on Z-Domain Autodisplay.

A regenerable immunoaffinity layer comprising covalently immobilized orientation-controlled antibodies was developed for use in a surface plasmon resonance (SPR) biosensor. For antibody orientation control, antibody-binding Z-domain-autodisplaying Escherichia coli (E. coli) cells and their outer membrane (OM) were utilized, and a disuccinimidyl crosslinker was employed for covalent antibody binding. To fabricate the regenerable immunoaffinity layer, capture antibodies were bound to autodisplayed Z-domains, and then treated with the crosslinker for chemical fixation to the Z-domains. Various crosslinkers, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and poly (ethylene glycol)-ylated bis (sulfosuccinimidyl)suberate (BS(PEG)5), were evaluated, and DSS at a concentration of 500 µM was confirmed to be optimal. The E. coli-cell-based regenerable HRP immunoassay was evaluated employing three sequential HRP treatment and regeneration steps. Then, the Oms of E. coli cells were isolated and layered on a microplate and regenerable OM-based HRP immunoassaying was evaluated. Five HRP immunoassays with four regeneration steps were found to be feasible. This regenerable, covalently immobilized, orientation-controlled OM-based immunoaffinity layer was applied to an SPR biosensor, which was capable of quantifying C-reactive protein (CRP). Five regeneration cycles were repeated using the demonstrated immunoaffinity layer with a signal difference of <10%.

Sesquiterpene Lactones with Dual Inhibitory Activity against the Trypanosoma brucei Pteridine Reductase 1 and Dihydrofolate Reductase.

The parasite Trypanosoma brucei (T. brucei) is responsible for human African trypanosomiasis (HAT) and the cattle disease "Nagana" which to this day cause severe medical and socio-economic issues for the affected areas in Africa. So far, most of the available treatment options are accompanied by harmful side effects and are constantly challenged by newly emerging drug resistances. Since trypanosomatids are auxotrophic for folate, their pteridine metabolism provides a promising target for an innovative chemotherapeutic treatment. They are equipped with a unique corresponding enzyme system consisting of the bifunctional dihydrofolate reductase-thymidylate synthase (TbDHFR-TS) and the pteridine reductase 1 (TbPTR1). Previously, gene knockout experiments with PTR1 null mutants have underlined the importance of these enzymes for parasite survival. In a search for new chemical entities with a dual inhibitory activity against the TbPTR1 and TbDHFR, a multi-step in silico procedure was employed to pre-select promising candidates against the targeted enzymes from a natural product database. Among others, the sesquiterpene lactones (STLs) cynaropicrin and cnicin were identified as in silico hits. Consequently, an in-house database of 118 STLs was submitted to an in silico screening yielding 29 further virtual hits. Ten STLs were subsequently tested against the target enzymes in vitro in a spectrophotometric inhibition assay. Five compounds displayed an inhibition over 50% against TbPTR1 as well as three compounds against TbDHFR. Cynaropicrin turned out to be the most interesting hit since it inhibited both TbPTR1 and TbDHFR, reaching IC50 values of 12.4 µM and 7.1 µM, respectively.

Natural Compounds Isolated from Stachybotrys chartarum Are Potent Inhibitors of Human Protein Kinase CK2.

A large number of secondary metabolites have been isolated from the filamentous fungus Stachybotrys chartarum and have been described before. Fourteen of these natural compounds were evaluated in vitro in the present study for their inhibitory activity towards the cancer target CK2. Among these compounds, stachybotrychromene C, stachybotrydial acetate and acetoxystachybotrydial acetate turned out to be potent inhibitors with IC50 values of 0.32 µM, 0.69 µM and 1.86 µM, respectively. The effects of these three compounds on cell proliferation, growth and viability of MCF7 cells, representing human breast adenocarcinoma as well as A427 (human lung carcinoma) and A431 (human epidermoid carcinoma) cells, were tested using EdU assay, IncuCyte® live-cell imaging and MTT assay. The most active compound in inhibiting MCF7 cell proliferation was acetoxystachybotrydial acetate with an EC50 value of 0.39 µM. In addition, acetoxystachybotrydial acetate turned out to inhibit the growth of all three cell lines completely at a concentration of 1 µM. In contrast, cell viability was impaired only moderately, to 37%, 14% and 23% in MCF7, A427 and A431 cells, respectively.

A modified flavonoid accelerates oligodendrocyte maturation and functional remyelination.

Myelination delay and remyelination failure following insults to the central nervous system (CNS) impede axonal conduction and lead to motor, sensory and cognitive impairments. Both myelination and remyelination are often inhibited or delayed due to the failure of oligodendrocyte progenitor cells (OPCs) to mature into myelinating oligodendrocytes (OLs). Digestion products of the glycosaminoglycan hyaluronan (HA) have been implicated in blocking OPC maturation, but how these digestion products are generated is unclear. We tested the possibility that hyaluronidase activity is directly linked to the inhibition of OPC maturation by developing a novel modified flavonoid that functions as a hyaluronidase inhibitor. This compound, called S3, blocks some but not all hyaluronidases and only inhibits matrix metalloproteinase activity at high concentrations. We find that S3 reverses HA-mediated inhibition of OPC maturation in vitro, an effect that can be overcome by excess recombinant hyaluronidase. Furthermore, we find that hyaluronidase inhibition by S3 accelerates OPC maturation in an in vitro model of perinatal white matter injury. Finally, blocking hyaluronidase activity with S3 promotes functional remyelination in mice with lysolecithin-induced demyelinating corpus callosum lesions. All together, these findings support the notion that hyaluronidase activity originating from OPCs in CNS lesions is sufficient to prevent OPC maturation, which delays myelination or blocks remyelination. These data also indicate that modified flavonoids can act as selective inhibitors of hyaluronidase activity and can promote OPC maturation, making them excellent candidates to accelerate myelination or promote remyelination following perinatal and adult CNS insults.

Targeting HSP90 dimerization via the C terminus is effective in imatinib-resistant CML and lacks the heat shock response.

Heat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34+CD38-) as well as the leukemic bulk (CD34+CD38+) of primary CML and in tyrosine kinase inhibitor (TKI)-resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.

Design, synthesis and biological evaluation of new embelin derivatives as CK2 inhibitors.

A new series of furan embelin derivatives was synthesized and characterized as ATP-competitive CK2 inhibitors. The new compounds were efficiently synthesized using a multicomponent approach from embelin (1), aldehydes and isonitriles through a Knoevenagel condensation/Michael addition/heterocyclization. Several compounds with inhibitory activities in the low micromolar or even submicromolar were identified. The most active derivative was compound 4l (2-(tert-butylamino)-3-(furan-3-yl)-5-hydroxy-6-undecylbenzofuran-4,7-dione) with an IC50 value of 0.63 µM. It turned out to be an ATP competitive CK2 inhibitor with a Ki value determined to be 0.48 µM. Docking studies allowed the identification of key ligand-CK2 interactions, which could help to further optimize this family of compounds as CK2 inhibitors.

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.

Purification-independent immunoreagents obtained by displaying nanobodies on bacteria surface.

The availability of preimmune libraries of antibody fragments allows for the fast generation of binders which can be expressed in both eukaryotic and prokaryotic systems. We exploited the recombinant nature of antibody fragments to demonstrate the possibility of expressing them as functional proteins displayed on the surface of Escherichia coli and by such a way to generate living reagents ready-to-use for diagnostics. Such immunoreagents were effectively exploited without the necessity of any purification step to prepare immunocapture surfaces suitable for the diagnostic of both cancer cells and toxic microalgae. The same nanobody-displaying bacteria were also engineered to coexpress GFP in their cytoplasm. Suspensions of such living fluorescent immunoreagents effectively bound to eukaryotic cells making them visible and quantifiable by flow cytometry analysis and using 96-well plate readers. The collected data showed the suitability of such living immunoreagents for reproducible and inexpensive diagnostic applications.

Cryptotanshinone from Salvia miltiorrhiza Roots Reduces Cytokeratin CK1/10 Expression in Keratinocytes by Activation of Peptidyl-prolyl-cis-trans-isomerase FKBP1A.

Cryptotanshinone (CTS) (1 µM) from the roots of Salvia miltiorrhiza exerts a strong influence on the terminal differentiation of human keratinocytes (HaCaT cell line, primary natural human keratinocytes) and downregulates the expression of differentiation-specific cytokeratins CK1 and CK10 on protein and gene level. Other differentiation specific proteins as involucrin, filaggrin, loricrin, and transglutaminase were not affected to a higher extent. CTS (1 µM) did not influence the cell viability and the proliferation of keratinocytes. Using a combination of drug affinity response target stability assay in combination with a proteomic approach and multivariate statistics for target elucidation, peptidyl-prolyl-cis-trans-isomerase FKBP1A (known target of inhibitors such as tacrolimus or rapamycin) was addressed as potential molecular target of CTS. The interaction of CTS with FKBP1A was additionally shown by thermal shift and enzymatic activity assays. Interestingly, CTS served as an activator of FKBP1A, which led to a reduced activity of the TGFß receptor pathway and therefore to a diminished CK1 and CK10 expression. The combination of the FKBP1A activator CTS with the inhibitor tacrolimus neutralized the effects of both compounds. From these data, a potential dermatological use of CTS and CTS-containing plant extracts (e.g., hydroalcoholic extract from the roots of S. miltiorrhiza) for keratinopathic ichthyosis, a disease characterized by overexpression of CK1 and CK10, is discussed. This study displays an experimental strategy for combining phytochemical aspects on active natural products with systematic identification of molecular targets on gene, protein, and cell level.

In Vitro and in Silico Evaluation of Bikaverin as a Potent Inhibitor of Human Protein Kinase CK2.

Protein kinase CK2 is an emerging target for therapeutic intervention in human diseases, particularly in cancer. Inhibitors of this enzyme are currently in clinical trials, indicating the druggability of human CK2. By virtual screening of the ZINC database, we found that the natural compound bikaverin can fit well in the ATP binding site of the target enzyme CK2. By further in vitro evaluation using CK2 holoenzyme, bikaverin turned to be a potent inhibitor with an IC50 value of 1.24 µM. In this work, the cell permeability of bikaverin was determined using a Caco-2 cell permeability assay as a prerequisite for cellular evaluation and the compound turned out to be cell permeable with a Papp- value of 4.46 × 10-6 cm/s. Bikaverin was tested for its effect on cell viability using a MTT assay and cell proliferation using an EdU assay in different cancer cell lines (MCF7, A427 and A431 cells). Cell viability and cell proliferation were reduced dramatically after treatment with 10 µM bikaverin for 24 h. Additionally the IncuCyte® live-cell imaging system was applied for monitoring the cytotoxicity of bikaverin in the three tested cancer cell lines. Finally, molecular dynamic studies were performed to clarify the ligand binding mode of bikaverin at the ATP binding site of CK2 and to identify the amino acids involved.