Inhibition of CREB phosphorylation by conjugates of adenosine analogues and arginine-rich peptides, inhibitors of PKA catalytic subunit.

Bisubstrate inhibitors of protein kinases associate simultaneously with two substrate-binding sites of the kinase and thus potentially possess better inhibitory potency and selectivity than inhibitors binding to only the conserved ATP-site of the kinase. We have previously used conjugates of adenosine analogues and arginine-rich peptides (ARCs) to develop proteolytically stable cell plasma membrane-permeable bisubstrate inhibitors whose biochemical affinities towards several basophilic protein kinases of the AGC group are in the picomolar range. The potency of bisubstrate inhibitors to affect the phosphorylation of proteins in living cells has been described in a limited number of publications.

Selective bisubstrate inhibitors with sub-nanomolar affinity for protein kinase Pim-1.

Potent and selective: The unique nature of the ATP binding pocket structure of Pim family protein kinases (PKs) was used for the development of bisubstrate inhibitors and a fluorescent probe with sub-nanomolar affinity. Conjugates of arginine-rich peptides with two ATP mimetic scaffolds were synthesized and tested as inhibitors of Pim-1. Against a panel of 124 protein kinases, a novel ARC-PIM conjugate selectively inhibited PKs of the Pim family.

Responsive microsecond-lifetime photoluminescent probes for analysis of protein kinases and their inhibitors.

Responsive ARC-Lum probes were used for measurement of the concentration of active protein kinases (PKs) and determination of affinity of inhibitors of PKs. ARC-Lum probes incorporate thiophene or a selenophene heterocycle and a fluorophore conjugated to the lysine residue in the peptide fragment. In the complex with a PK, ARC-Lum probes emit long-lifetime (microsecond-scale) luminescence at the emission wavelengths of the fluorescent label if the complex is illuminated at the excitation wavelength of the thiophene- or selenophene-containing phosphorescence donors.

Time-gated luminescence microscopy with responsive nonmetal probes for mapping activity of protein kinases in living cells.

A photoluminescence probe ARC-1185, possessing both high affinity towards basophilic protein kinases (PKs) and microsecond-scale luminescence lifetime when associated with a kinase, was used for the mapping of ARC-1185-PK complexes in living cells with time-gated luminescence microscopy.

Conjugates of 5-isoquinolinesulfonylamides and oligo-D-arginine possess high affinity and selectivity towards Rho kinase (ROCK).

In the present work, conjugates of 5-isoquinolinesulfonylamides and D-arginine-rich peptides were developed into highly potent inhibitors for basophilic protein kinases. Based on Hidaka's inhibitor H9, a generic fluorescent probe ARC-1083 was constructed possessing subnanomolar dissociation constant towards several kinases of the AGC-group. Thereafter, Hidaka's inhibitor HA1077 or Fasudil was conjugated with oligo-D-arginine resulting in the compound ARC-3002 revealing high affinity towards ROCK-II (K(d)=20 pM) and over 160-fold selectivity compared to PKAc.

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.

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.

Small-molecule FRET probes for protein kinase activity monitoring in living cells.

In this study, the applicability of fluorescently labeled adenosine analogue-oligoarginine conjugates (ARC-Photo probes) for monitoring of protein kinase A (PKA) activity in living cells was demonstrated. ARC-Photo probes possessing subnanomolar affinity towards the catalytic subunit of PKA (PKAc) and competitive with the regulatory subunit (PKAr), penetrate cell plasma membrane and associate with PKAc fused with yellow fluorescent protein (PKAc-YFP). Detection of inter-molecular Förster resonance energy transfer (FRET) efficiency between the fluorophores of the fusion protein and ARC-Photo probe can be used for both the evaluation of non-labeled inhibitors of PKAc and for monitoring of cAMP signaling via detection of changes in the activity of PKA as a cAMP downstream effector.

Adenosine analogue-oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIalpha).

Introduction: Type I cGMP-dependent protein kinase (PKGIalpha) belongs to the family of cyclic nucleotide-dependent protein kinases and is one of the main effectors of cGMP. PKGIalpha is involved in regulation of cardiac contractility, vasorelaxation, and blood pressure; hence, the development of potent modulators of PKGIalpha would lead to advances in the treatment of a variety of cardiovascular diseases.

Aim: Representatives of ARC-type compounds previously characterized as potent inhibitors and high-affinity fluorescent probes of PKA catalytic subunit (PKAc) were tested towards PKGIalpha to determine that ARCs could serve as activity regulators and sensors for the latter protein kinase both in vitro and in complex biological systems.

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.

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.

Carbocyclic 3'-deoxyadenosine-based highly potent bisubstrate-analog inhibitor of basophilic protein kinases.

Carbocyclic analogs of 3'-deoxyadenosine were synthesized as racemates and the resulting stereoisomers were separated by chromatography on a chiral column. The conjugation of obtained compounds with hexa-(D-arginine) via 6-aminohexanoic acid linker led to a highly potent inhibitor of several basophilic protein kinases with some selectivity towards cAMP-dependent protein kinase.

Surface-plasmon-resonance-based biosensor with immobilized bisubstrate analog inhibitor for the determination of affinities of ATP- and protein-competitive ligands of cAMP-dependent protein kinase.

Interactions between adenosine-oligoarginine conjugates (ARC), bisubstrate analog inhibitors of protein kinases, and catalytic subunits of cAMP-dependent protein kinase (cAPK Calpha) were characterized with surface-plasmon-resonance-based biosensors. ARC-704 bound to the immobilized kinase with subnanomolar affinity. The immobilization of ARC-704 to the chip surface via streptavidin-biotin complex yielded a high-affinity surface (K(D)=16nM).

Conjugation of adenosine and hexa-(D-arginine) leads to a nanomolar bisubstrate-analog inhibitor of basophilic protein kinases.

Conjugates of oligoarginine peptides with adenine, adenosine, adenosine-5'-carboxylic acid, and 5-isoquinolinesulfonic acid were synthesized and characterized as bisubstrate-analog inhibitors of cAMP-dependent protein kinase. Adenosine and adenine derivatives were connected to the N- or C-terminus of peptides containing four to six L- or D-arginine residues via a linker with a length that had been optimized in structure-activity studies. The orientation of the peptide chain strongly affected the activity of compounds incorporating D-arginines.

Fluorometric TLC assay for evaluation of protein kinase inhibitors.

A fluorometric assay for measuring protein kinase activity has been developed. The assay is based on the separation of fluorescently marked substrate 5-carboxytetramethylrhodamine-kemptide (5-TAMRA-kemptide) from its phosphorylated counterpart by TLC and quantification of the product ratiometrically by fluorescence imaging. The utility of the assay was demonstrated by measuring the activity of cAMP-dependent protein kinase.