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.

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. RESULTS: Structure-activity profiling of ARCs with PKGIalpha in vitro demonstrated both similarities as well as differences to corresponding profiling with PKAc, whereas ARC-903 and ARC-668 revealed low nanomolar displacement constants and inhibition IC(50) values with both cyclic nucleotide-dependent kinases. The ability of ARC-based fluorescent probes to penetrate cell plasma membrane was demonstrated in the smooth muscle tissue of rat cerebellum isolated arteries, and the compound with the highest affinity in vitro (ARC-903) showed also potential for in vivo applications, fully abolishing the PKG1alpha-induced vasodilation.

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-oligoarginine conjugate, a novel bisubstrate inhibitor, effectively dissociates the actin cytoskeleton.

Aberrant regulation of protein kinases impairs normal cellular functioning and may lead to disease. The protein kinase involved in the regulation of the dynamics of the actin cytoskeleton, Rho-kinase (ROCK), phosphorylates various substrates (e.g. myosin light chain, myosin phosphatase), causing the formation of actin fibers and tension inside cells. Hyperactivation of ROCK, for example, causes hypertension and cardiovascular disorders. Thus, the design of highly specific protein kinase inhibitors is of the utmost importance. To date, the majority of inhibitors investigated have been found to mimic and compete with ATP. However, in the present study we characterized the cellular effects of a novel bisubstrate inhibitor -- adenosine-oligoarginine conjugate (ARC) -- designed to interfere simultaneously with the ATP site and the substrate-binding pocket of basophilic kinases. ARC effectively pulled down ROCK from cell lysates, showed no cytotoxicity and suppressed the assembly of the actin cytoskeleton (especially central actin bundles) as the result of interference with the activity of the kinase. Combination of ARC with chloroquine yielded a stronger inhibitory effect and gave results similar to treatment with Y-27632. However, treatment with ARC produced more actin fragments and yielded a longer-lasting effect than treatment with Y-27632. Additionally, quantification of phosphorylated myosin light chain levels in ARC-treated or Y-27632-treated cells implies that ARC is more effective than Y-27632 in suppressing the phosphorylation of at least one of the substrates of ROCK. We believe that the described bisubstrate strategy could be a useful lead for designing novel, highly specific inhibitors for different protein kinases.

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. The biligand inhibitor containing Hidaka's H9 isoquinolinesulfonamide connected to the L-peptide had 65 times higher potency than the corresponding adenosine-containing conjugate, while both types of the conjugate comprising D-peptides had similar low nanomolar activity. Two of the most active adenosine- and H9-peptide conjugates were tested in the panel of 52 different kinases. At 1 microM concentration, both compounds showed strong (more than 95%) inhibition of several basophilic AGC kinases, including pharmaceutically important kinases ROCK II and PKB/Akt.

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.

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). The bisubstrate character of ARC-704 was demonstrated with various ligands targeted to ATP-binding pocket (ATP and inhibitors H89 and H1152P) and protein-substrate-binding domain of Calpha (RIIalpha and GST-PKIalpha) in competition assays. The experiments performed on surfaces with different immobilization levels of ARC-704 produced similar results. The closeness of the obtained affinities of the tested compounds to the inhibitory potencies and affinities of the compounds measured with other methods demonstrates the applicability of the chip with the immobilized biligand inhibitor for the characterization of both ATP- and substrate protein-competitive ligands of basophilic protein kinases.