Hans-gottfried Genieser - Academia.edu (original) (raw)

Papers by Hans-gottfried Genieser

BMC Pharmacology, Aug 1, 2011

Tetrahedron Letters, 1988

ABSTRACT

European journal of pharmacology, Oct 1, 1994

In the present study, the inhibitory effect of the cGMP analog (Rp)-8-(para-chlorophenylthio)guan... more In the present study, the inhibitory effect of the cGMP analog (Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate ((Rp)-8-pCPT-cGMPS) on the cGMP-dependent protein kinase-mediated protein phosphorylation in intact human platelets was investigated. In vitro phosphorylation experiments with the substrate kemptide demonstrated an inhibition of the cGMP-dependent protein kinase by (Rp)-8-pCPT-cGMPS with a g i of 0.5 /zM. In intact human platelets, (Rp)-8-pCPT-cGMPS antagonized the activation of the cGMP-dependent protein kinase by 8-pCPT-cGMP without affecting cAMP-dependent protein kinase or cGMP-regulated phosphodiesterases. The data obtained suggest that (Rp)-8-pCPT-cGMPS may be a useful tool for studying the role of cGMP in vitro and in intact cells.

Developmental Biology, May 1, 1989

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methylad... more Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Cyclic AMP seems to play a negative role in maturation since 1-MeAde triggers a decrease of the oocyte cAMP concentration and since intracellular microinjections of cAMP delay or inhibit maturation. Cyclic GMP is also inhibitory but other nucleotides such as cCMP, cIMP, and cUMP are inactive. The involvement of cAMP and cGMP in the control of oocyte maturation has been further investigated by the use of the stereoisomers of the phosphodiesterase-stable adenosine and guanosine 3',5'-phosphorothioates (cAMPS and cGMPS). The Sp isomers of cAMPS and cGMPS respectively activate cAMP-dependent protein kinase and cGMP-dependent kinase, while the Rp isomers inhibit the kinases. Extracellular addition of these cAMPS and cGMPS isomers has no effect on the oocytes. Intracellular microinjection of the kinase-activating (Sp)-cAMPS and (Sp)-cGMPS delays or inhibits 1-MeAde-induced maturation in a concentration-dependent manner (I50, 30 and 300 microM, respectively). Microinjections of (Rp)-cAMPS and (Rp)-cGMPS have no inhibitory effects and neither trigger nor facilitate maturation. Using various analogs, we found that the delaying or inhibiting effect is restricted to the compounds activating cAMP-dependent kinase, while the compounds inactive on or inhibiting the kinase have no effects on maturation. The inhibitory effect of (Sp)-cAMPS can be reversed by comicroinjection of the heat-stable inhibitor of cAMP-dependent protein kinase, by comicroinjection of the antagonist (Rp)-cAMPS, or by an increase in the 1-MeAde concentration. The negative effects of (Sp)-cAMPS or (Sp)-cGMPS are observed only when these isomers are microinjected during the hormone-dependent period. These results suggest that a cAMP-dependent inhibitory pathway participates in the maintenance of the prophase arrest of oocytes and that 1-MeAde acts both by inhibiting this negative pathway (dis-inhibitory pathway) and by stimulating a parallel activatory pathway leading to oocyte maturation. The generality of this mechanism is discussed.

Proteomics, Mar 1, 2008

Functional proteomics aims to describe cellular protein networks in depth based on the quantifica... more Functional proteomics aims to describe cellular protein networks in depth based on the quantification of molecular interactions. In order to study the interaction of adenosine‐3′,5′‐cyclic monophosphate (cAMP), a general second messenger involved in several intracellular signalling networks, with one of its respective target proteins, the regulatory (R) subunit of cAMP dependent protein kinase (PKA), a number of different methods was employed. These include fluorescence polarisation (FP), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), amplified luminescence proximity homogeneous assay (ALPHA‐screen), radioligand binding or activity‐based assays. Kinetic, thermodynamic and equilibrium binding data of a variety of cAMP derivatives to several cAMP binding domains were integrated in a single database system, we called KinetXBase, allowing for very distinct data formats. KinetXBase is a practical data handling system for molecular interaction data of any kind, providing a synopsis of data derived from different technologies. This supports ongoing efforts in the bioinformatics community to devise formal concepts for a unified representation of interaction data, in order to enable their exchange and easy comparison. KinetXBase was applied here to analyse complex cAMP binding data and highly site‐specific cAMP analogues could be identified. The software package is free for download by academic users.

ChemBioChem, Sep 1, 2008

Synthesis of 8-(4-Chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate, acetoxymethy... more Synthesis of 8-(4-Chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate, acetoxymethyl ester (8-pCPT-2'-O-Me-cAMP-AM): Synthesis was performed as described previously with some minor modifications. [S1] 360 µmol 8-pCPT-2'-O-Me-cAMP, diisopropylethylammonium salt, were suspended in 1000 µL DMF. After addition of 1800 µmol (180 µL; 5 equiv) acetoxymethyl bromide and 1080 µmol (185 µL; 3 equiv) diisopropylethylamine, the reaction mixture was stirred at ambient temperature for 30 minutes. Progress of AM-ester formation was monitored by analytical HPLC with 40% acetonitrile as eluent. The reaction was stopped by evaporation of all volatile components in a speed-vac centrifuge under reduced pressure with oil pump vacuum. The residues were re-dissolved in 0.8 mL DMF, and purified by preparative HPLC using 40% acetonitrile as eluent. The product-containing fractions were collected and evaporated. 110 µmol 8-pCPT-2'-O-Me-cAMP-AM were isolated as mixture of axial and equato rial isomers with a purity of >97% (yield: 30.6%). Small amounts of pure isomers for NMR experiments were generated by repeated analytical HPLC runs.

Journal of Biological Chemistry, Sep 1, 2003

Little is known about the relative role of cAMP-dependent protein kinase (cAPK) and guanine excha... more Little is known about the relative role of cAMP-dependent protein kinase (cAPK) and guanine exchange factor directly activated by cAMP (Epac) as mediators of cAMP action. We tested cAMP analogs for ability to selectively activate Epac1 or cAPK and discriminate between the binding sites of Epac and of cAPKI and cAP-KII. We found that commonly used cAMP analogs, like 8-Br-cAMP and 8-pCPT-cAMP, activate Epac and cAPK equally as well as cAMP, i.e. were full agonists. In contrast, 6-modified cAMP analogs, like N 6-benzoyl-cAMP, were inefficient Epac activators and full cAPK activators. Analogs modified in the 2-position of the ribose induced stronger Epac1 activation than cAMP but were only partial agonists for cAPK. 2-O-Alkyl substitution of cAMP improved Epac/cAPK binding selectivity 10-100fold. Phenylthio substituents in position 8, particularly with MeO-or Cl-in p-position, enhanced the Epac/cAPK selectivity even more. The combination of 8-pCPT-and 2-O-methyl substitutions improved the Epac/cAPK binding selectivity about three orders of magnitude. The cAPK selectivity of 6-substituted cAMP analogs, the preferential inhibition of cAPK by moderate concentrations of Rp-cAMPS analogs, and the Epac selectivity of 8-pCPT-2-O-methyl-cAMP was also demonstrated in intact cells. Using these compounds to selectively modulate Epac and cAPK in PC-12 cells, we observed that analogs selectively activating Epac synergized strongly with cAPK specific analogs to induce neurite outgrowth. We therefore conclude that cAMP-induced neurite outgrowth is mediated by both Epac and cAPK.

Apple Academic Press eBooks, Apr 15, 2011

Background: A novel fluorescent cAMP analog (8-[Pharos-575]-adenosine-3', 5'-cyclic monophosphate... more Background: A novel fluorescent cAMP analog (8-[Pharos-575]-adenosine-3', 5'-cyclic monophosphate) was characterized with respect to its spectral properties, its ability to bind to and activate three main isoenzymes of the cAMP-dependent protein kinase (PKA-Iα, PKA-IIα, PKA-IIβ) in vitro, its stability towards phosphodiesterase and its ability to permeate into cultured eukaryotic cells using resonance energy transfer based indicators, and conventional fluorescence imaging. Results: The Pharos fluorophore is characterized by a Stokes shift of 42 nm with an absorption maximum at 575 nm and the emission peaking at 617 nm. The quantum yield is 30%. Incubation of the compound to RIIα and RIIβ subunits increases the amplitude of excitation and absorption maxima significantly; no major change was observed with RIα. In vitro binding of the compound to RIα subunit and activation of the PKA-Iα holoenzyme was essentially equivalent to cAMP; RII subunits bound the fluorescent analog up to ten times less efficiently, resulting in about two times reduced apparent activation constants of the holoenzymes compared to cAMP. The cellular uptake of the fluorescent analog was investigated by cAMP indicators. It was estimated that about 7 μM of the fluorescent cAMP analog is available to the indicator after one hour of incubation and that about 600 μM of the compound had to be added to intact cells to half-maximally dissociate a PKA type IIα sensor. Conclusion: The novel analog combines good membrane permeability-comparable to 8-Br-cAMP-with superior spectral properties of a modern, red-shifted fluorophore. GFP-tagged regulatory subunits of PKA and the analog co-localized. Furthermore, it is a potent, PDE-resistant activator of PKA-I and-II, suitable for in vitro applications and spatial distribution evaluations in living cells.

BMC Chemical Biology, Feb 12, 2009

Background: In the eukaryotic cell the cAMP-dependent protein kinase (PKA) is a key enzyme in sig... more Background: In the eukaryotic cell the cAMP-dependent protein kinase (PKA) is a key enzyme in signal transduction and represents the main target of the second messenger cAMP. Here we describe the design, synthesis and characterisation of specifically tailored cAMP analogs which can be utilised as a tool for affinity enrichment and purification as well as for proteomics based analyses of cAMP binding proteins. Results: Two sets of chemical binders were developed based on the phosphorothioate derivatives of cAMP, Sp-cAMPS and Rp-cAMPS acting as cAMP-agonists and-antagonists, respectively. These compounds were tested via direct surface plasmon resonance (SPR) analyses for their binding properties to PKA R-subunits and holoenzyme. Furthermore, these analogs were used in an affinity purification approach to analyse their binding and elution properties for the enrichment and improvement of cAMP binding proteins exemplified by the PKA R-subunits. As determined by SPR, all tested Sp-analogs provide valuable tools for affinity chromatography. However, Sp-8-AEA-cAMPS displayed (i) superior enrichment properties while maintaining low unspecific binding to other proteins in crude cell lysates, (ii) allowing mild elution conditions and (iii) providing the capability to efficiently purify all four isoforms of active PKA R-subunit in milligram quantities within 8 h. In a chemical proteomics approach both sets of binders, Rp-and Sp-cAMPS derivatives, can be employed. Whereas Sp-8-AEA-cAMPS preferentially binds free R-subunit, Rp-AHDAA-cAMPS, displaying antagonist properties, not only binds to the free PKA R-subunits but also to the intact PKA holoenzyme both from recombinant and endogenous sources. Conclusion: In summary, all tested cAMP analogs were useful for their respective application as an affinity reagent which can enhance purification of cAMP binding proteins. Sp-8-AEA-cAMPS was considered the most efficient analog since Sp-8-AHA-cAMPS and Sp-2-AHA-cAMPS, demonstrated incomplete elution from the matrix, as well as retaining notable amounts of bound protein contaminants. Furthermore it could be demonstrated that an affinity resin based on Rp-8-AHDAA-cAMPS provides a valuable tool for chemical proteomics approaches.

M 8-[ϕ-575]-cAMP for 30 minutes. Cells were fixed and fluorescence was imaged using confocal micr... more M 8-[ϕ-575]-cAMP for 30 minutes. Cells were fixed and fluorescence was imaged using confocal microscopy: (a,c) green fluorescence of GFP (b,e), red fluorescence of 8-[ϕ-575]-cAMP, (c,f) merged images. The scale bar indicates 10 μm.<b>Copyright information:</b>Taken from "Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog"http://www.biomedcentral.com/1471-2091/9/18BMC Biochemistry 2008;9():18-18.Published online 25 Jun 2008PMCID:PMC2443153.

Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP... more Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog

Die Erfindung betrifft neue Verbindungen der Formeln (X) und (I), die die Interaktion von Protein... more Die Erfindung betrifft neue Verbindungen der Formeln (X) und (I), die die Interaktion von Proteinkinase A (PKA) und Proteinkinase A-Ankerproteinen (AKAP) modulieren, insbesondere inhibieren. Die Erfindung betrifft weiterhin pharmazeutische Mittel, insbesondere fur die Behandlung von Krankheiten, die mit einer Storung des cAMP Signalweges assoziiert sind, insbesondere Krankheiten wie Hypertrophie des Herzens, koronare Herzkrankheiten, Hypertonie und Herzinsuffizienz.

Take-down policy If you believe that this document breaches copyright please contact us providing... more Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Proceedings of the National Academy of Sciences of the United States of America, Jan 27, 2018

Inherited retinal degeneration (RD) is a devastating and currently untreatable neurodegenerative ... more Inherited retinal degeneration (RD) is a devastating and currently untreatable neurodegenerative condition that leads to loss of photoreceptor cells and blindness. The vast genetic heterogeneity of RD, the lack of "druggable" targets, and the access-limiting blood-retinal barrier (BRB) present major hurdles toward effective therapy development. Here, we address these challenges () by targeting cGMP (cyclic guanosine- 3',5'-monophosphate) signaling, a disease driver common to different types of RD, and () by combining inhibitory cGMP analogs with a nanosized liposomal drug delivery system designed to facilitate transport across the BRB. Based on a screen of several cGMP analogs we identified an inhibitory cGMP analog that interferes with activation of photoreceptor cell death pathways. Moreover, we found liposomal encapsulation of the analog to achieve efficient drug targeting to the neuroretina. This pharmacological treatment markedly preserved in vivo retinal func...

European Journal of Biochemistry, 1989

cAMP analogs, all 96 of which were modified in the adenine moiety, were examined quantitatively f... more cAMP analogs, all 96 of which were modified in the adenine moiety, were examined quantitatively for their ability to inhibit the binding of [3H]cAMP to each of the two classes (A and B) of CAMP-binding sites of type I (rabbit skeletal muscle) and type I1 (bovine heart) CAMP-dependent protein kinase. The study showed that analogs can be constructed that have a higher affinity than cAMP for a binding site. N6-phenyl-CAMP had 18fold increased affinity for site A of RI (AI) and 40-fold increased affinity for site AIL 2-ch!oro-S-methylarnino-cAMP had a 7-fold increased affinity for BI, and 8-(4-~hloropheny!thio)-cAMP had 17-fold increased affinity for BII. Analogs could discriminate between the two classes of binding sites by more than two orders of magnitude in binding affinity: 2-chloro-8-methylamino-CAMP had 170-fold higher affinity for BI than for AI, and 2-n-butyl-8-thiobenzyl-CAMP had 700-fold higher affinity for BII than for AIL Analogs could also discriminate between the homologous binding sites of the isozymes: 2-n-butyl-8-bromo-CAMP had 260-fold higher affinity for A1 than for A11 (22-fold higher for BII than BI), and 8-piperidino-CAMP had 50-fold higher affinity for BII than for BI (and 50-fold higher for A1 than for AII). The data suggest the following conclusions. (a) Stacking interactions are important for the binding of cAMP to all the binding sites. (b) Subtle differences exist between the sites as to the optimal electron distribution in the adenine ring since modifications that withdraw electrons at C2 and donate at C8 favour binding to BE, and disfavour binding to A1 and AII. (c) There are no hydrogen bonds between the adenine ring of cAMP and any of the binding sites. (d) All sites bind cAMP in the syn conformation. (e) The subsites adjacent to the N6 and C8 positions may have nonpolar neighbouring regions since hydrophobic substituents at N6 could increase the affinity for A1 and A11 and similar substituents at C8 could increase the affinity for €311. Finally, (0 the sites differed in their ability to accomodate bulky substituents at C2 and C8. For all compounds tested, their potency as activators of protein kinases I and I1 was found to correlate, in a predictable fashion, to their mean affinity for the two classes of binding sites, rather than to the affinity for only one of the sites. The major receptor for cAMP in mammalian cells is the regulatory subunit of CAMP-dependent protein kinase [1-31. Two general types (cAKI and cAKII) of CAMP-dependent protein kinases have been distinguished based on their order

Cell death & disease, 2011

The IPC-81 cell line is derived from the transplantable BNML model of acute myelogenic leukemia (... more The IPC-81 cell line is derived from the transplantable BNML model of acute myelogenic leukemia (AML), known to be a reliable predictor of the clinical efficiency of antileukemic agents, like the first-line AML anthracycline drug daunorubicin (DNR). We show here that cAMP acted synergistically with DNR to induce IPC cell death. The DNR-induced death differed from that induced by cAMP by (1) not involving Bim induction, (2) being abrogated by GSK3β inhibitors, (3) by being promoted by the HSP90/p23 antagonist geldanamycin and truncated p23 and (4) by being insensitive to the CRE binding protein (CREB) antagonist ICER and to cyclin-dependent protein kinase (CDK) inhibitors. In contrast, the apoptosis induced by cAMP correlated tightly with Bim protein expression. It was abrogated by Bim (BCL2L11) downregulation, whether achieved by the CREB antagonist ICER, by CDK inhibitors, by Bim-directed RNAi, or by protein synthesis inhibitor. The forced expression of BimL killed IPC-81(WT) cells...

Proceedings of the National Academy of Sciences, 1994

In t(15;17) acute promyelocytic leukemia, all-trans retinoic acid (RA) induces leukemic cell matu... more In t(15;17) acute promyelocytic leukemia, all-trans retinoic acid (RA) induces leukemic cell maturation in vitro and remission in acute promyelocytic leukemia patients, but in vivo treatments invariably lead to relapse with resistance to RA. NB4, a maturation-inducible cell line, and NB4-RAr sublines (R1 and R2) displaying no maturation in the presence of RA have been isolated from a patient in relapse. We show that resistance to maturation is not a mere unresponsiveness to RA: rather, R1 "resistant" cells do respond to RA (1 microM) by sustained growth, become competent to undergo terminal maturation, and up-regulate CD11c/CD18 integrins. Interestingly, maturation of "resistant" cells, rendered competent by RA, can be achieved by cAMP-elevating agents (prostaglandin E, isoproterenol, cholera toxin, or phosphodiesterase inhibitor) or stable agonistic cAMP analogs such as (SP)-8-chloroadenosine cyclic 3',5'-phosphorothioate. This shows that activation of c...