Darja Lavogina

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.

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.

Diversity of bisubstrate binding modes of adenosine analogue-oligoarginine conjugates in protein kinase a and implications for protein substrate interactions.

Crystal structures of the catalytic subunit α of cAMP-dependent protein kinase (PKAc) with three adenosine analogue-oligoarginine conjugates (ARCs) are presented. The rationally designed ARCs include moieties that, in combination, target both the ATP- and the peptide-substrate-binding sites of PKAc, thereby taking advantage of high-affinity binding interactions offered by the ATP site while utilizing an additional mechanism for target specificity via binding to the peptide substrate site. The crystal structures demonstrate that, in accord with the previously reported bisubstrate character of ARCs, the inhibitors occupy both binding sites of PKAc. Further, they show new binding modes that may also apply to natural protein substrates of PKAc, which have not been revealed by previous crystallographic studies. The crystal structures described here contribute to the understanding of the substrate-binding patterns of PKAc and should also facilitate the design of inhibitors targeting PKAc and related protein kinases.

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 Hidakas inhibitor H9, a generic fluorescent probe ARC-1083 was constructed possessing subnanomolar dissociation constant towards several kinases of the AGC-group. Thereafter, Hidakas 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.

Long residence times revealed by Aurora A kinase-targeting fluorescent probes derived from inhibitors MLN8237 and VX-689.

We report the development of three fluorescent probes for protein kinase Aurora A that are derived from the well‐known inhibitors MLN8237 and VX‐689 (MK‐5108). Two of these probes target the ATP site of Aurora A, and one targets simultaneously the ATP and substrate sites of the kinase. The probes were tested in an assay with fluorescence polarisation/anisotropy readout, and we demonstrated slow association kinetics and long residence time of the probes (kon 105–107 M−1 s−1, koff 10−3–10−4 s−1; residence time 500–3000 s). The presence of the Aurora A activator TPX2 caused a significant reduction in the on‐rate and increase in the off‐rate of fluorescent probes targeting ATP site. These observations were supported by Aurora A inhibition assays with MLN8237 and VX‐689. Overall, our results emphasise the importance of rational design of experiments with these compounds and correct interpretation of the obtained data.

Acetoxymethyl Ester of Tetrabromobenzimidazole–Peptoid Conjugate for Inhibition of Protein Kinase CK2 in Living Cells

CK2 is a ubiquitous serine/threonine protein kinase, which has the potential to catalyze the generation of a large proportion of the human phosphoproteome. Due to its role in numerous cellular functions and general anti-apoptotic activity, CK2 is an important target of research with therapeutic potential. This emphasizes the need for cell-permeable highly potent and selective inhibitors and photoluminescence probes of CK2 for investigating the protein phosphorylation networks in living cells. Previously, we had developed bisubstrate inhibitors for CK2 (CK2-targeted ARCs) that showed remarkable affinity (KD < 1 nM) and selectivity, but lacked proteolytic stability and plasma membrane permeability. In this report, the structures of CK2-targeted ARCs were modified for the application in live cells. Based on structure-activity studies, proteolytically stable achiral oligoanionic peptoid conjugates of 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz) were constructed. Affinity of the conjugates toward CK2 reached subnanomolar range. Acetoxymethyl (AM) prodrug strategy was applied for loading TBBz-peptoid conjugates into living cells. The uptake of inhibitors was visualized by live cell imaging and the reduction of the phosphorylation levels of two CK2-related phosphosites, Cdc37 pSer13 and NFκB pSer529, was demonstrated by Western blot analysis.

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

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.

Bisubstrate inhibitor approach for targeting mitotic kinase Haspin.

During the past decade, the basophilic atypical kinase Haspin has emerged as a key player in mitosis responsible for phosphorylation of Thr3 residue of histone H3. Here, we report the construction of conjugates comprising an aromatic fragment targeted to the ATP-site of Haspin and a peptide mimicking the N-terminus of histone H3. The combination of effective solid phase synthesis procedures and a high throughput binding/displacement assay with fluorescence anisotropy readout afforded the development of inhibitors with remarkable subnanomolar affinity toward Haspin. The selectivity profiles of novel conjugates were established by affinity studies with a model basophilic kinase (catalytic subunit of cAMP-dependent protein kinase) and by a commercial 1-point inhibition assay with 43 protein kinases.

Selective Biligand Inhibitor of CK2 Increases Caspase-3 Activity in Cancerous Cells and Inhibits Platelet Aggregation

Cancer cells express high levels of CK2, and its inhibition leads to apoptosis. CK2 has therefore emerged as a new drug target for cancer therapy. A biligand inhibitor ARC‐772 was constructed by conjugating 4‐(2‐amino‐1,3‐thiazol‐5‐yl)benzoic acid and a carboxylate‐rich peptoid. ARC‐772 was found to bind CK2 with a Kd value of 0.3 nm and showed remarkable CK2 inhibitory selectivity in a panel of 140 protein kinases (Gini coefficient: 0.75 at c=100 nm). ARC‐775, the acetoxymethyl ester prodrug of ARC‐772, was efficiently taken up by cells. Once internalized, the inhibitor is activated by cellular esterase activity. In HeLa cancer cells ARC‐775 was found to activate caspase‐3 (an apoptosis marker) at sub‐micromolar concentrations (EC50=0.3 μm), a 20‐fold lower extracellular concentration than CX‐4945, the only CK2 inhibitor under clinical trials. At micromolar concentrations, ARC‐775 was also found to inhibit ADP‐induced aggregation of human platelets. The overall results of this study demonstrate that oligo‐anionic biligand inhibitors have good potential for drug development.