Asko Uri

Bisubstrate Inhibitors of Protein Kinases: from Principle to Practical Applications

Bisubstrate inhibitors consist of two conjugated fragments, each targeted to a different binding site of a bisubstrate enzyme. The design of bisubstrate inhibitors presupposes the formation of the ternary complex in the course of the catalyzed reaction. The principle advantage of bisubstrate inhibitors is their ability to generate more interactions with the target enzyme that could result in improved affinity and selectivity of the conjugates, when compared with single‐site inhibitors. Among phosphotransferases, the approach was first successfully used for adenylate kinase in 1973. Since then, several types of bisubstrate inhibitors have been developed for protein kinases, including conjugates of peptides with nucleotides, adenosine derivatives and potent ATP‐competitive inhibitors. Earlier bisubstrate inhibitors had pharmacokinetic qualities that were unsuitable for cellular experiments and hence were mostly used for in vitro studies. The recently constructed conjugates of adenosine derivatives and D‐arginine‐rich peptides (ARCs) possess high kinase affinity, high biological and chemical stability and good cell plasma membrane penetrative properties that enable their application in the regulation of cellular protein phosphorylation balances in cell and tissue experiments.

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 G proteins 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 G proteins ≈ 500‐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.

2-Alkylthio-substituted platelet P2Y12 receptor antagonists reveal pharmacological identity between the rat brain Gi-linked ADP receptors and P2Y12

The Gi-linked platelet ADP receptor, now designated as P2Y12, accounts for ADP-induced inhibition of adenylyl cyclase in platelets and certain clonal rat cell lines. The pharmacology of this receptor is well characterized. Based on the functional approach of [35S]GTPgammaS autoradiography, we recently disclosed the widespread presence of Gi-linked ADP receptors in the rat nervous system. Based on initial pharmacological analysis, these receptors were strikingly similar with P2Y12. Here, we extend this analysis by comparing the potencies of six 2-alkylthio-substituted ATP analogues, including the adenosine-aspartate conjugate 2-hexylthio-AdoOC(O)Asp2 and five AR-C compounds (AR-C67085, AR-C69931, AR-C78511, AR-C69581, AR-C70300) with wide range of affinities towards P2Y12, in reversing 2-methylthio-ADP stimulated G protein activity in rat brain sections and human platelet membranes. Closely matching pIC50 values (r2=0.99) revealed pharmacological similarity between the two receptors with one exception: AR-C67085 more avidly recognized the platelet P2Y12. Further analysis of the rat brain pIC50 data against those available for three of the AR-C compounds in reversing P2Y12-mediated adenylyl cyclase inhibition in rat platelets (r2=0.96) and rat C6 glioma cells (r2=1.00) demonstrated that the three P2Y receptors are pharmacologically indistinguishable. We conclude that the rat brain Gi-linked ADP receptors, as revealed using [35S]GTPgammaS autoradiography, correspond to P2Y12.

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.

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.

Time-gated luminescence assay using nonmetal probes for determination of protein kinase activity-based disease markers.

A novel nonmetal optical probe ARC-1063 whose long-lifetime luminescence is induced by association with the target protein kinase is used for the measurement of the concentration of catalytic subunit of protein kinase A (PKAc) in complicated biological solutions. High affinity (K(D) = 10 pM toward PKAc) and unique optical properties of the probe enable its application for the measurement of picomolar concentrations of PKAc in the presence of high concentrations of other proteins. The described assay is applicable in the high-throughput format with the instrument setups designed for lanthanide-based time-gated (time-resolved) luminescence methods. The assay is used for demonstration that extracellular PKAc (ECPKA) is present in plasma samples of all healthy persons and cancer patients but great care must be taken for procedures of treatment of blood samples to avoid disruption, damage, or activation of platelets in the course of plasma (or serum) preparation and conservation.

Bisubstrate fluorescent probes and biosensors in binding assays for HTS of protein kinase inhibitors

Conjugates of adenosine mimics and d-arginine-rich peptides (ARCs) are potent inhibitors of protein kinases (PKs) from the AGC group. Labeling ARCs with fluorescent dyes or immobilizing on chip surfaces gives fluorescent probes (ARC-Photo) and biosensors that can be used for high-throughput screening (HTS) of inhibitors of protein kinases. The bisubstrate character (simultaneous association with both binding sites of the kinase) and high affinity of ARCs allow ARC-based probes and sensors to be used for characterization of inhibitors targeted to either binding site of the kinase with affinities in whole nanomolar to micromolar range. The ability to penetrate cell plasma membrane and bind to the target kinase fused with a fluorescent protein leads to the possibility to use ARC-Photo probes for high content screening (HCS) of inhibitors in cellular milieu with detection of intensity of Förster resonance energy transfer (FRET) between two fluorophores.

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.