Gerda Raidaru

High-affinity bisubstrate probe for fluorescence anisotropy binding/displacement assays with protein kinases PKA and ROCK

The bisubstrate fluorescent probe ARC-583 (Adc-Ahx-(D-Arg)(6)-d-Lys(5-TAMRA)-NH2) and its application for the characterization of both ATP- and protein/peptide substrate-competitive inhibitors of protein kinases PKA (cyclic AMP-dependent protein kinase) and ROCK (rho kinase) in fluorescence polarization-based assay are described. High affinity of the probe (K(D)=0.48 nM toward PKA) enables its application for the characterization of inhibitors with nanomolar and micromolar potency and determination of the active concentration of the kinase in individual experiments as well as in the high-throughput screening format. The probe can be used for the assessment of protein-protein interactions (e.g., between regulatory and catalytic subunits of PKA) and as a cyclic AMP biosensor.

[35S]GTPγS autoradiography reveals a wide distribution of Gi/o-linked ADP receptors in the nervous system : close similarities with the platelet P2YADP receptor

No Gi‐linked P2Y receptors have been cloned to date but the presence of such receptors is thought to be restricted to platelets and certain clonal cell lines. Using the functional approach of [35S]guanosine 5′‐[γ‐thio]‐triphosphate autoradiography, we uncovered the widespread presence of such receptors in the CNS. Under conditions in which the prominent signal due to tonic adenosine receptor activity is masked, ADP and ATP stimulated G‐protein activity in multiple grey and white matter regions. Localization in the grey matter suggests inhibitory auto‐/heteroreceptor function. In the white matter, activated Gu2003proteins appeared as ‘hot spots’ (presumed oligodendrocyte progenitors) with scattered distribution along the main fibre tracts. Responses to ATP were diminished under conditions that inhibited degradation, suggesting that prior conversion to ADP explained agonist action. Uracil nucleotides were ineffective but 2‐methylthio‐ADP activated Gu2003proteins ≈u200a500‐fold more potently than ADP, although both were similarly degraded. Throughout the brain, ADP‐dependent G‐protein activity was reversed by 2‐hexylthio‐AdoOC(O)Asp2, a non‐phosphate ATP analogue, whereas selective P2Y1 receptor antagonists proved ineffective. A similar receptor was also disclosed from the adrenal medulla. These data witness a hitherto unrecognized abundance of Gi/o‐linked ADP receptors in the nervous system. Biochemical and pharmacological behaviour suggests striking similarities to the elusive platelet P2YADP receptor.

Adenosine 5'-carboxylic acid peptidyl derivatives as inhibitors of protein kinases

A new class of protein kinase bisubstrate-analog inhibitors was designed on the basis of adenosine-5-carboxylic acid derivatives, where a short peptide was attached to the 5-carbon atom of the adenosine sugar moiety via a linker chain. The potency and selectivity of these inhibitors were adjusted by relevant combination of these structural fragments, resembling the structure of the bisubstrate complex of the peptide phosphorylation reaction.

Structural Analysis of ARC-Type Inhibitor (ARC-1034) Binding to Protein Kinase A Catalytic Subunit and Rational Design of Bisubstrate Analogue Inhibitors of Basophilic Protein Kinases

The crystal structure of a complex of the catalytic subunit (type alpha) of cAMP-dependent protein kinase (PKA C alpha) with ARC-type inhibitor (ARC-1034), the presumed lead scaffold of previously reported adenosine-oligo-arginine conjugate-based (ARC-type) inhibitors, was solved. Structural elements important for interaction with the kinase were established with specifically modified derivatives of the lead compound. On the basis of this knowledge, a new generation of inhibitors, conjugates of adenosine-4-dehydroxymethyl-4-carboxylic acid moiety and oligo(D-arginine), was developed with inhibitory constants well into the subnanomolar range. The structural determinants of selectivity of the new compounds were established in assays with ROCK-II and PKBgamma.

Protein-induced long lifetime luminescence of nonmetal probes.

Time-resolved luminometry-based assays have great potential for measurements in complicated biological solutions and living cells as the measured signal can be easily distinguished from nanosecond lifetime background fluorescence of organic compounds and autofluorescence of cells. In the present study we discovered that binding of a thiophene- or a selenophene-containing heteroaromatic moiety (luminescence donor) to the purine-binding pocket of a protein kinase (PK) induces long lifetime photoluminescence signal that is largely intensified through efficient energy transfer to a fluorescent dye present in close proximity to the luminescence donor. The developed ARC-Lum probes possessing 19-266 μs luminescence lifetime when associated with the target kinase can be used for determination of activity of basophilic PKs, characterization of inhibitors of PKs, and as cAMP sensors. An ARC-Lum probe was also used for the determination of kinetic parameters of inhibitor binding to the catalytic subunit of protein kinase A (PKAc). Effective real-time monitoring of the activation of PKA by Forskolin and the displacement of an ARC-Lum probe from its complex with PKA by inhibitor H89 was performed in live cells. The discovered phenomenon, protein-induced long lifetime luminescence of aromatic probes is very likely to occur with all PKs and many other proteins.

Characterization of glucose oxidase immobilization onto mica carrier by atomic force microscopy and kinetic studies

Glucose oxidase (E.C 1.1.3.4) immobilized onto activated surface of mica was analyzed by enzymatic kinetics and visualization with atomic force microscopy (AFM). The activity of the immobilized enzyme decreased with the decrease of concentration of gamma-aminopropyltrimethoxysilane used for the first step of activation of mica, while AFM analysis showed similar homogeneous filling of the surface with the enzyme. The comparison of enzyme activity with its surface filling revealed that there has to be additional vertical structures, which cannot be visualized by the methods of AFM. The simultaneous decrease of the silanizing agent and the concentration of the enzyme led to molecular resolution for the enzyme on the surface of mica. This allows to propose the described method also for analyzing other surfaces of solid materials with coupled biomolecules.

Liquid-phase synthesis of a pegylated adenosine–oligoarginine conjugate, cell-permeable inhibitor of cAMP-dependent protein kinase

An adenosine-oligoarginine conjugate (ARC) was assembled in a stepwise manner on a poly(ethylene glycol) carrier. The pegylated conjugate inhibited cAMP-dependent protein kinase with IC(50)=460 nM and the cellular uptake of its BODIPY FL derivative was demonstrated and compared to that of free ARC with fluorescence microscopy.

A subnanomolar fluorescent probe for protein kinase CK2 interaction studies

Up-regulation of an acidophilic protein kinase, CK2, has been established in several types of cancer. This cognition has made CK2 an important target for drug development for cancer chemotherapy. The characterization of potential drug candidates, determination of the structure and clarification of the functions of CK2 could be facilitated by the application of small-molecule fluorescent probes that bind to the active site of the enzyme with high affinity and selectivity. We have used a bisubstrate approach for the development of a highly potent inhibitor of CK2. 4,5,6,7-Tetrabromo-1H-benzimidazole was conjugated with peptides containing multiple aspartate residues via different linkers. The design of the inhibitors was by crystallographic analysis of the complex of an inhibitor with the catalytic subunit of the enzyme (CK2α). The inhibitory potency of the synthesized compounds was established in a kinetic assay that used thin layer chromatography for the measurement of the rate of phosphorylation of fluorescently labelled peptide 5-TAMRA-RADDSDDDDD. The most potent inhibitor, ARC-1502 (K(i) = 0.5 nM), revealed high selectivity for CK2α in a panel of 140 protein kinases. Labelling of ARC-1502 with PromoFluor-647 gave the fluorescent probe ARC-1504 that possessed subnanomolar affinity towards both CK2α and the holoenzyme. The probe was used in a fluorescence anisotropy-based binding assay to measure the concentration of CK2α and characterize non-labelled ligands binding to the active site of CK2α.

Identification of the ability of highly charged nanomolar inhibitors of protein kinases to cross plasma membranes and carry a protein into cells

A fluorescently labeled adenosine-oligoarginine conjugate (ARC), nanomolar bisubstrate analogue-type inhibitor of basophilic protein kinases PKA and PKC, readily enters cells of different origin and localizes into cytoplasm and nucleus. Moreover, the biotinylated derivative of ARC is able to deliver avidin, a non-covalently attached protein cargo, into cells.

D2 dopamine receptor-G protein coupling. Cross-regulation of agonist and guanosine nucleotide binding sites

The cross regulation of agonist binding to D2 dopamine receptors and guanosine nucleotide binding to G proteins was studied using membranes of Chinese hamster ovary cells expressing rat D2short dopamine receptors. All guanosine nucleotides studied caused a concentration-dependent loss of high-affinity agonist binding sites of D2 receptors with potencies corresponding to their affinity to bind to G proteins in these membranes. On the other hand, the dopaminergic agonists, but not antagonists, decreased the affinities of guanosine diphosphate and guanosine monophosphate, but not of guanosine 5-(gamma-thiotriphosphate). The cross regulation of ligand binding to D2 dopamine receptors and G proteins suggests the existence of several conformational states of these proteins during the signal transduction and that the high-affinity state of agonist binding is a transient state of the agonist-receptor-G protein complex, where no nucleotides are bound.