Bivalent ligands, including bisubstrate inhibitors, are conjugates of pharmacophores, which simultaneously target two binding sites of the biomolecule. Such structures offer attainable means for the development of compounds whose ability to bind to the biological target could be modulated by an external trigger. In the present work, two deactivatable bisubstrate inhibitors of basophilic protein kinases (PKs) were constructed by conjugating the pharmacophores via linkers that could be cleaved in response to external stimuli. The inhibitor ARC-2121 incorporated a photocleavable nitrodibenzofuran-comprising β-amino acid residue in the structure of the linker. The pharmacophores of the other deactivatable inhibitor ARC-2194 were conjugated via reduction-cleavable disulfide bond. The disassembly of the inhibitors was monitored by HPLC-MS. The affinity and inhibitory potency of the inhibitors toward cAMP-dependent PK (PKAcα) were established by an equilibrium competitive displacement assay and enzyme activity assay, respectively. The deactivatable inhibitors possessed remarkably high 1–2-picomolar affinity toward PKAcα. Irradiation of ARC-2121 with 365 nm UV radiation led to reaction products possessing a 30-fold reduced affinity. The chemical reduction of ARC-2194 resulted in the decrease of affinity of over four orders of magnitude. The deactivatable inhibitors of PKs are valuable tools for the temporal inhibition or capture of these pharmacologically important enzymes.
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.
In the lead generation phase of drug development, the screening and characterization of new bioactive compounds, pharmacological hits, are of crucial importance [1]. The quality of these research a...
Recent clinical success with targeted covalent inhibitors points to new possibilities for development of protein kinase (PK)-targeted drugs by exploiting reactive cysteine residues in and around the ATP-binding site. However, more than 300 human PKs lack cysteine residues in the ATP-binding site. Here, we report the first covalent bisubstrate PK inhibitor whose electrophilic warhead reaches outside the ATP-binding site and reacts with a distant cysteine residue. A series of covalent inhibitors and their reversible counterparts were synthesized and characterized. The most potent reversible inhibitor possessed picomolar affinity and its cysteine-reactive counterpart revealed high value of kinact/KI ratio (6.2 × 107 M–1 s–1) for the reaction with the catalytic subunit of cAMP-dependent PK (PKAc). Under optimized conditions, fluorescent dye-labeled covalent inhibitors demonstrated PKA-selectivity in the cell lysate and reacted with several proteins inside live cells, including PKAc. The disclosed compounds serve as leads for targeting PKs possessing an analogously positioned cysteine residue.
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