Bifunctional Ligands for Inhibition of Tight-Binding Protein-Protein Interactions.

The acknowledged potential of small-molecule therapeutics targeting disease-related protein-protein interactions (PPIs) has promoted active research in this field. The strategy of using small molecule inhibitors (SMIs) to fight strong (tight-binding) PPIs tends to fall short due to the flat and wide interfaces of PPIs. Here we propose a biligand approach for disruption of strong PPIs.

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.

Fluorescent photoaffinity probes for mitotic protein kinase Aurora A.

We combined the advantages of the selective inhibitor VX689, the bisubstrate-analogue conjugate approach, and photoreactive amino acids to develop 8 photoaffinity probes for Aurora A. The most efficient compounds possessed one-digit nanomolar KD values in the equilibrium binding assay, inhibited Aurora A at elevated concentrations of ATP in the phosphorylation assay in the presence of TPX2, and formed covalent complexes with the recombinant kinase or Aurora A in HeLa cells upon UV-irradiation. The recognition of the correct target by the probes during formation of the covalent complex in the biochemical assay and in situ was demonstrated by competition experiments using the non-labelled inhibitors VX689 and MLN8237.

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.

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.

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.

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.

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.

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.

Effect of the structure of adenosine mimic of bisubstrate-analog inhibitors on their activity towards basophilic protein kinases.

Previously reported structural fragments that associate with the ATP-binding pocket of basophilic protein kinases were conjugated with d-arginine-containing peptides. Inhibitory potency of the resulting bisubstrate-analog inhibitors towards PKA and ROCK-II extended to subnanomolar range. The conjugates incorporating 2-pyrimidyl-5-amidothiophene fragment had the highest activity and at 100 nM concentration exhibited over 80% inhibition of most of the tested basophilic kinases of the AGC group.

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.

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.

Synthesis of potential purinoceptor antagonists: application of P1-tBU phosphazene base for alkylation of adenine in solution and on solid phase.

Alkylation of adenine in solution and on solid phase was accelerated by phosphazene base P1-tBu compared to mineral bases. The reactions in solution afforded regioselectively the appropriate N9-alkylated adenines with high preparative yields while the reaction with polystyrene resin-bound N-bromoacetylated peptides gave three regioisomers (alkylated at the N9, N7, and N3 position of adenine) in a 4:2:1 molar ratio. Ten novel nonphosphate nucleotide analogues were tested in an ADP-induced platelet aggregation assay.

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.

Kinetic analysis of inhibition of cAMP-dependent protein kinase catalytic subunit by the peptide-nucleoside conjugate AdcAhxArg6.

Kinetic analysis of the inhibition of the phosphorylation of Kemptide, (LRRASLG), catalyzed by the catalytic subunit of cAMP-dependent protein kinase, by a peptide-nucleoside conjugate inhibitor AdcAhxArg6 was carried out over a wide range of ATP and peptide concentrations. A simple procedure was proposed for characterization of the interaction of this inhibitor with the free enzyme, and with the enzyme-ATP and enzyme-peptide complexes. The second-order rate constants, calculated from the steady-state reaction kinetics, were used for this analysis to avoid the complications related to the complex catalytic mechanism of the protein kinase catalyzed reaction.

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.

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

Glucose oxidase (E.C 1.1.

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.