Protein kinase CK2 (casein kinase 2) mainly exists as heterotetrameric holoenzyme with two catalytic subunits (CK2α or CK2α′) bound to a homodimer of non-catalytic subunits (CK2β). With CSNK2A1 and CSNK2A2 , the human genome contains two paralogs encoding catalytic CK2 subunits. Both gene products, called CK2α and CK2α′, strongly interact with CK2β. An earlier report that CK2α′ has a lower CK2β affinity than CK2α is confirmed via isothermal titration calorimetry in this study. Furthermore, we show with a fluorescence-anisotropy assay that a CK2β-competitive peptide binds less strongly to CK2α′ than to CK2α. The reason for the reduced affinity of CK2α′ to CK2β and CK2β competitors is puzzling: both isoenzymes have identical amino acid compositions at their CK2β interfaces, but the β4β5 loop, a component of this interface, is conformationally less adaptable in CK2α′ than in CK2α due to intramolecular constraints. To release these constraints, we constructed a CK2α′ mutant that was equalized to CK2α at the backside of the β4β5 loop. Concerning thermostability, affinity to CK2β or CK2β competitors and 3D-structure next to the β4β5 loop, this CK2α′ mutant is more similar to CK2α than to its own wild-type, suggesting a critical role of the β4β5 loop adaptability for CK2β affinity.

The serine/threonine kinase CK2 (formerly known as casein kinase II) plays a crucial role in various CNS disorders and is highly expressed in various types of cancer. Therefore, inhibiting this key kinase could be promising for the treatment of these diseases. The CK2 holoenzyme is formed by the recruitment of two catalytically active CK2α and/or CK2α′ subunits by a regulatory CK2β dimer. Starting with the lead furocarbazole W16 (4) inhibiting the CK2α/CK2β interaction, analogous pyrrolocarbazoles were prepared and tested for their protein–protein interaction inhibition (PPII). The key step of the synthesis was a multicomponent Levy reaction of 2-(indolyl)acetate 6, benzaldehydes 7, and N-substituted maleimides 8. Targeted modifications were performed by the saponification of the tetracyclic ester 9a, followed by the coupling of the resulting acid 10 with diverse amines. The replacement of the O-atom of the lead furocarbazole 4 by an N-atom in pyrrolocarbazoles retained or even increased the inhibition of the CK2α/CK2β interaction. The large benzyloxazolidinyl moiety of 4 could be replaced by smaller N-substituents without the loss of the PPII. The introduction of larger substituents at the 2-position and/or at p-position of the phenyl moiety at the 10-position to increase the surface for the inhibition of the PPI did not enhance the inhibition of the CK2α/CK2β association. The strong inhibition of the CK2α/CK2β association by the histidine derivative (+)-20a (Ki = 6.1 µM) translated into a high inhibition of the kinase activity of the CK2 holoenzyme (CK2α2β2, IC50 = 2.5 µM). Thus, 20a represents a novel lead compound inhibiting CK2 via the inhibition of the association of the CK2α and Ck2β subunits.

In this work, we designed, synthesized, and evaluated two types of multineurotargeting compounds using a pharmacophore merging strategy, aiming to develop potential treatments for Alzheimer's disease. We combined belinostat, an FDA-approved unselective histone deacetylase (HDAC) inhibitor, with the 5-substituted indole core of contilisant, known for its antioxidant and neuroprotective properties as well as its potent inhibitory activity against monoamine oxidases (MAOs), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). Among these, compounds 8c (HDAC1, IC50 = 0.019 μM; HDAC6, IC50 = 0.040 μM; AChE, IC50 = 20.06 μM; BChE, IC50 = 17.10 μM; MAO-B, IC50 = 2.14 μM), and 9c (HDAC1, IC50 = 0.126 μM; HDAC6, IC50 = 0.020 μM; AChE, IC50 = 2.73 μM; BChE, IC50 = 4.03 μM; MAO-B, IC50 = 1.18 μM) emerged as the most promising candidates. These compounds warrant further investigation as potential treatments for Alzheimer's disease due to their unique inhibition profiles and favorable mode of inhibition.

G proteins are interacting partners of G protein-coupled receptors (GPCRs) in eukaryotic cells. Upon G protein activation, the ability of the Gα subunit to exchange GDP for GTP determines the intracellular signal transduction. Although various studies have successfully shown that both Gαs and Gαi have an opposite effect on the intracellular cAMP production, with the latter being commonly described as "more active", the functional analysis of Gαs is a comparably more complicated matter. Additionally, the thorough investigation of the ubiquitously expressed variants of Gαs, Gαs(short) and Gαs(long), is still pending. Since the previous experimental evaluation of the activity and function of the Gαs isoforms is not consistent, the focus was laid on structural investigations to understand the GTPase activity. Herein, we examined recombinant human Gαs by applying an established methodological setup developed for Gαi characterization. The ability for GTP binding was evaluated with fluorescence and fluorescence anisotropy assays, whereas the intrinsic hydrolytic activity of the isoforms was determined by a GTPase assay. Among different nucleotide probes, BODIPY FL GTPγS exhibited the highest binding affinity towards the Gαs subunit. This work provides a deeper understanding of the Gαs subunit and provides novel information concerning the differences between the two protein variants.

The triplet-sensitized (by the solvent acetone) as well as the direct (lambda ex=300-320 nm) photochemical decarboxylation of N-phthaloylated gamma-aminobutyric acid (GABA) derivatives are versatile and high-yielding routes to benzopyrrolizidines via intramolecular electron transfer initiated decarboxylation followed by radical coupling. The ss-mono- and ss,ss'-disubstituted N-phthaloyl GABA derivatives 7 a-7 g, respectively, were applied as substrates. Decarboxylative photocyclization yielded hydroxy benzopyrrolizidines 8 a-8 g in high chemical yields and with moderate diastereoselectivities from the ss-monosubstituted substrates. The analogous alpha-substituted GABA derivatives 11 a-11 c were also applied as potential substrates for memory of chirality effects. The reaction quantum yields of the photodecarboxylation reactions for the parent GABA derivative 13 and for the new substrates 7 h and 11 a were determined by the quantum yield determination system (QYDS) and showed a remarkable concentration dependency indicating aggregation at higher substrate concentrations. Inhibition studies on the atherogenic human serine hydrolase cholesterol esterase showed derivatives 8 a and 8 d to exhibit a hyperbolic mode of inhibition with moderate IC50 values of about 60-80 mu M. Reagent and catalyst-free conversion of numerous GABA derivatives into biologically active pyrroloisoindolinones is achieved by direct or triplet-sensitized photolyses in aqueous solvent mixtures with high quantum yields. image

Introduction Subtotal petrosectomy is an established treatment for chronic otitis. However, the wound closure is to be regarded as a critical step which often determines the success or failure of the surgical intervention. Impaired wound healing in the area of the external auditory canal or of the retroauricular incision is a frequent complication. We describe a modification of the surgical technique ("split flap") that has significantly reduced the complication rate in our department.

Ziel/Aim The aim of this work is to couple radioisotopes of iodine to two selected estrogen derivatives in order to investigate their estrogen-receptor (ER) mediated cell binding to urogenital tumors. The long-term goal is the development of a radiopharmaceutical for receptor-mediated diagnosis and therapy of urogenital carcinomas in men.

The preparation of 24 estrogens, their estrogen receptor (ER) affinity and studies of radioiodinated estrogen binding to ER-positive male bladder tumor cells (HTB9) are described. The estrogens with the highest affinity were selected using fluorescence anisotropy assays. A 2,2,2-trifluoroethyl group at the 11 beta-position caused particularly promising affinity. (Radio)iodination was performed on the 17 alpha-vinyl group. Binding studies on HTB9 cells revealed picomolar affinities of radioconjugates 19 and 31, indicating promising ability for targeting of urogenital tumors.

Einleitung Die subtotale Petrosektomie ist als sanierende Prozedur bei chronischer Otitis etabliert. Der Wundverschluss ist jedoch als kritischer Schritt anzusehen und entscheidet oft über Erfolg oder Misserfolg der Operation. Die Wundheilungsstörung im Bereich von Gehörgang oder retroaurikulärer Narbe ist eine häufige Komplikation. Wir beschreiben eine Modifikation der OP-Technik („Split-Flap“), die in unserer Abteilung die Komplikationsrate deutlich gesenkt hat.

In addition to the classic functions of proteins, such as acting as a biocatalyst or binding partner, the conformational states of proteins and their remodeling upon stimulation need to be considered. A prominent example of a protein that undergoes comprehensive conformational remodeling is transglutaminase 2 (TGase 2), the distinct conformational states of which are closely related to particular functions. Its involvement in various pathophysiological processes, including fibrosis and cancer, motivates the development of theranostic agents, particularly based on inhibitors that are directed toward the transamidase activity. In this context, the ability of such inhibitors to control the conformational dynamics of TGase 2 emerges as an important parameter, and methods to assess this property are in great demand. Herein, we describe the application of the switchSENSE® principle to detect conformational changes caused by three irreversibly binding Nε-acryloyllysine piperazides, which are suitable radiotracer candidates of TGase 2. The switchSENSE® technique is based on DNA levers actuated by alternating electric fields. These levers are immobilized on gold electrodes with one end, and at the other end of the lever, the TGase 2 is covalently bound. A novel computational method is introduced for describing the resulting lever motion to quantify the extent of stimulated conformational TGase 2 changes. Moreover, as a complementary biophysical method, native polyacrylamide gel electrophoresis was performed under similar conditions to validate the results. Both methods prove the occurrence of an irreversible shift in the conformational equilibrium of TGase 2, caused by the binding of the three studied Nε-acryloyllysine piperazides.

Gα proteins as part of heterotrimeric G proteins are molecular switches essential for G protein-coupled receptor- mediated intracellular signaling. The role of the Gα subunits has been examined for decades with various guanine nucleotides to elucidate the activation mechanism and Gα protein-dependent signal transduction. Several approaches describe fluorescent ligands mimicking the GTP function, yet lack the efficient estimation of the proteins' GTP binding activity and the fraction of active protein. Herein, we report the development of a reliable fluorescence anisotropy-based method to determine the affinity of ligands at the GTP-binding site and to quantify the fraction of active Gαi1 protein. An advanced bacterial expression protocol was applied to produce active human Gαi1 protein, whose GTP binding capability was determined with novel fluorescently labeled guanine nucleotides acting as high-affinity Gαi1 binders compared to the commonly used BODIPY FL GTPγS. This study thus contributes a new method for future investigations of the characterization of Gαi and other Gα protein subunits, exploring their corresponding signal transduction systems and potential for biomedical applications.

Transglutaminase 2 (TGase 2) is a multifunctional protein which is involved in various physiological and pathophysiological processes. The latter also include its participation in the development and progression of malignant neoplasms, which are often accompanied by increased protein synthesis. In addition to the elucidation of the molecular functions of TGase 2 in tumor cells, knowledge of its concentration that is available for targeting by theranostic agents is a valuable information. Herein, we describe the application of a recently developed fluorescence anisotropy (FA)-based assay for the quantitative expression profiling of TGase 2 by means of transamidase-active enzyme in cell lysates. This assay is based on the incorporation of rhodamine B-isonipecotyl-cadaverine (R-I-Cad) into N,N-dimethylated casein (DMC), which results in an increase in the FA signal over time. It was shown that this reaction is not only catalyzed by TGase 2 but also by TGases 1, 3, and 6 and factor XIIIa using recombinant proteins. Therefore, control measurements in the presence of a selective irreversible TGase 2 inhibitor were mandatory to ascertain the specific contribution of TGase 2 to the overall FA rate. To validate the assay regarding the quality of quantification, spike/recovery and linearity of dilution experiments were performed. A total of 25 cancer and 5 noncancer cell lines were characterized with this assay method in terms of their activatable TGase 2 concentration (fmol/µg protein lysate) and the results were compared to protein synthesis data obtained by Western blotting. Moreover, complementary protein quantification methods using a biotinylated irreversible TGase 2 inhibitor as an activity-based probe and a commercially available ELISA were applied to selected cell lines to further validate the results obtained by the FA-based assay. Overall, the present study demonstrates that the FA-based assay using the substrate pair R-I-Cad and DMC represents a facile, homogenous and continuous method for quantifying TGase 2 activity in cell lysates.

Animal models and clinical studies suggest an influence of angiotensin II (AngII) on the pathogenesis of liver diseases via the renin-angiotensin system. AngII application increases portal blood pressure, reduces bile flow, and increases permeability of liver tight junctions. Establishing the subcellular localization of angiotensin II receptor type 1 (AT1R), the main AngII receptor, helps to understand the effects of AngII on the liver. We localized AT1R in situ in human and porcine liver and porcine gallbladder by immunohistochemistry. In order to do so, we characterized commercial anti-AT1R antibodies regarding their capability to recognize heterologous human AT1R in immunocytochemistry and on western blots, and to detect AT1R using overlap studies and AT1R-specific blocking peptides. In hepatocytes and canals of Hering, AT1R displayed a tram-track-like distribution, while in cholangiocytes AT1R appeared in a honeycomb-like pattern; i.e., in liver epithelia, AT1R showed an equivalent distribution to that in the apical junctional network, which seals bile canaliculi and bile ducts along the blood-bile barrier. In intrahepatic blood vessels, AT1R was most prominent in the tunica media. We confirmed AT1R localization in situ to the plasma membrane domain, particularly between tight and adherens junctions in both human and porcine hepatocytes, cholangiocytes, and gallbladder epithelial cells using different anti-AT1R antibodies. Localization of AT1R at the junctional complex could explain previously reported AngII effects and predestines AT1R as a transmitter of tight junction permeability.

Nowadays, several options are available to treat patients with conductive or mixed hearing loss. Whenever surgical intervention is not possible or contra-indicated, and amplification by a conventional hearing device (e.g., behind-the-ear device) is not feasible, then implantable hearing devices are an indispensable next option. Implantable bone-conduction devices and middle-ear implants have advantages but also limitations concerning complexity/invasiveness of the surgery, medical complications, and effectiveness. To counsel the patient, the clinician should have a good overview of the options with regard to safety and reliability as well as unequivocal technical performance data. The present consensus document is the outcome of an extensive iterative process including ENT specialists, audiologists, health-policy scientists, and representatives/technicians of the main companies in this field. This document should provide a first framework for procedures and technical characterization to enhance effective communication between these stakeholders, improving health care.

Cochlear variability is of key importance for the clinical use of cochlear implants, the most successful neuroprosthetic device that is surgically placed into the cochlear scala tympani. Despite extensive literature on human cochlear variability, few information is available on the variability of the modiolar wall. In the present study, we analyzed 108 corrosion casts, 95 clinical cone beam computer tomographies (CTs) and 15 µCTs of human cochleae and observed modiolar variability of similar and larger extent than the lateral wall variability. Lateral wall measures correlated with modiolar wall measures significantly. ~ 49% of the variability had a common cause. Based on these data we developed a model of the modiolar wall variations and related the model to the design of cochlear implants aimed for perimodiolar locations. The data demonstrate that both the insertion limits relevant for lateral wall damage (approximate range of 4–9 mm) as well as the dimensions required for optimal perimodiolar placement of the electrode (the point of release from the straightener; approximate range of 2–5mm) are highly interindividually variable. The data demonstrate that tip fold-overs of preformed implants likely result from the morphology of the modiolus (with radius changing from base to apex), and that optimal cochlear implantation of perimodiolar arrays cannot be guaranteed without an individualized surgical technique.

The antithrombotic prodrugs ticlopidine and clopidogrel are thienotetrahydro-pyridine derivatives that are metabolized in the liver to produce thiols that irreversibly block adenosine diphosphate (ADP)-activated P2Y12 receptors on thrombocytes. In their native, nonmetabolized form, both drugs were reported to act as inhibitors of ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39). CD39 catalyzes the extracellular hydrolysis of nucleoside tri- and diphosphates, mainly adenosine 5'-triphosphate (ATP) and ADP, yielding adenosine monophosphate, which is further hydrolyzed by ecto-5'-nucleotidase (CD73) to produce adenosine. While ATP has proinflammatory effects, adenosine is a potent anti-inflammatory, immunosuppressive agent. Inhibitors of CD39 and CD73 have potential as novel checkpoint inhibitors for the immunotherapy of cancer and infection. In the present study, we investigated 2-substituted thienotetrahydropyridine derivatives, structurally related to ticlopidine, as CD39 inhibitors. Due to their substituent on the 2-position, they will not be metabolically transformed into reactive thiols and can, therefore, be expected to be devoid of P2Y12 receptor-antagonistic activity in vivo. Several of the investigated 2-substituted thienotetrahydropyridine derivatives showed concentration-dependent inhibition of CD39. The most potent derivative, 32, showed similar CD39-inhibitory potency to ticlopidine, both acting as allosteric inhibitors. Compound 32 showed an improved selectivity profile: While ticlopidine blocked several NTPDase isoenzymes, 32 was characterized as a novel dual inhibitor of CD39 and CD73.

The human cochlea has a highly individual microanatomy. Cochlear implantation therefore requires an evaluation of the individual cochlear anatomy to reduce surgical risk of implantation trauma. However, in-vivo cochlear imaging is limited in resolution. To overcome this issue, cochlear models based on exact anatomical data have been developed. These models can be fitted to the limited parameters available from clinical imaging to provide a prediction of the precise cochlear microanatomy. Recently, models have become available with improved precision that additionally allow predicting the 3D form of an individual cochlea. The present study has further improved the precision of modelling by incorporating microscopic details of a large set of 108 human cochleae from corrosion casts. The new model provides a more flexible geometric shape that can better predict local variations like vertical dips and jumps and provides an approximation of frequency allocation in the cochlea. The outcome of this and five other models have been quantified (validated) on an independent set of 20 µCTs of human cochleae. The new model outperformed previous models and is freely available for download and use.

Human cochlear anatomy is highly variable. The phenomenon has been first described qualitatively, followed by a quantitative variability assessment with detailed anatomical models of the human cochlea. However, all previous work focused on lateral cochlear wall. Few information is available on the variability of the modiolar wall. Modiolar variability, likely determined by variability in the spiral ganglion, provides key information on when during ontogenesis the individual cochlear morphology is established: before and/or after neuronal structures are formed. In the present study we analyzed 108 corrosion casts, 95 clinical cone beam computer tomographies and 15 µCTs of human cochleae and observed modiolar variability of similar and larger extent than the lateral wall variability. Lateral wall measures correlated with modiolar wall measures significantly. ~49% of the variability has a common cause, very likely established already during the time when the spiral ganglion is formed. Proximity of other neuronal and vascular structures, defining the remaining variability in scalar spaces, are determined later in ontogenesis, when the scalae are formed. The present data further allows implications for perimodiolar cochlear implants and their tip fold-overs. In particular, the data demonstrate that tip fold-overs of preformed implants likely result from the morphology of the modiolus (with radius changing from base to apex), and that optimal cochlear implantation of perimodiolar arrays cannot be guaranteed without an individualized surgical technique.

The transamidase activity of transglutaminase 2 (TGase 2) is considered to be important for several pathophysiological processes including fibrotic and neoplastic tissue growth, whereas in healthy cells this enzymatic function is predominantly latent. Methods that enable the highly sensitive detection of TGase 2, such as application of radiolabeled activity-based probes, will support the exploration of the enzyme's function in various diseases. In this context, the radiosynthesis and detailed in vitro radiopharmacological evaluation of an 18F-labeled Nε-acryloyllysine piperazide are reported. Robust and facile detection of the radiotracer-TGase 2 complex by autoradiography of thin layer plates and polyacrylamide gels after chromatographic and electrophoretic separation owing to irreversible covalent bond formation was demonstrated for the isolated protein, cell lysates, and living cells. By use of this radiotracer, quantitative data on the expression profile of activatable TGase 2 in mouse organs and selected tumors were obtained for the first time by autoradiography of tissue sections.

A reliable solution-phase synthesis of the water-soluble dipeptidic fluorogenic transglutaminase substrate Z-Glu(HMC)-Gly-OH is presented. The route started from Z-Glu-OH, which was converted into the corresponding cyclic anhydride. This building block was transformed into the regioisomeric α- and γ-dipeptides. The key step was the esterification of Z-Glu-Gly-OtBu with 4-methylumbelliferone. The final substrate compound was obtained in an acceptable yield and excellent purity without the need of purification by RP-HPLC. The advantage of this acyl donor substrate for the kinetic characterisation of inhibitors and amine-type acyl acceptor substrates is demonstrated by evaluating commercially available or literature-known irreversible inhibitors and the biogenic amines serotonin, histamine and dopamine, respectively.