Binding assay for characterization of protein kinase inhibitors possessing sub-picomolar to sub-millimolar affinity

Sinijärv, Hedi; Wu, Shanshan; Ivan, Taavi; Laasfeld, Tõnis; Viht, Kaido; Uri, Asko

doi:10.1016/j.ab.2017.05.017

Cited by 16 publications

(18 citation statements)

References 42 publications

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“…The fluorescence anisotropy was calculated and plotted against the logarithm of the compound concentration and fit to the log inhibitor vs. response–variable slope (four parameters) equation in Prism (GraphPad). The IC 50 of the inhibitor was converted into K d as described in Sinijarv et al (2017) using the equation…”

Section: Methodsmentioning

confidence: 99%

A Covalent Calmodulin Inhibitor as a Tool to Study Cellular Mechanisms of K-Ras-Driven Stemness

Okutachi

Manoharan

Kiriazis

et al. 2021

Front. Cell Dev. Biol.

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Recently, the highly mutated oncoprotein K-Ras4B (hereafter K-Ras) was shown to drive cancer cell stemness in conjunction with calmodulin (CaM). We previously showed that the covalent CaM inhibitor ophiobolin A (OphA) can potently inhibit K-Ras stemness activity. However, OphA, a fungus-derived natural product, exhibits an unspecific, broad toxicity across all phyla. Here we identified a less toxic, functional analog of OphA that can efficiently inactivate CaM by covalent inhibition. We analyzed a small series of benzazulenones, which bear some structural similarity to OphA and can be synthesized in only six steps. We identified the formyl aminobenzazulenone 1, here named Calmirasone1, as a novel and potent covalent CaM inhibitor. Calmirasone1 has a 4-fold increased affinity for CaM as compared to OphA and was active against K-Ras in cells within minutes, as compared to hours required by OphA. Calmirasone1 displayed a 2.5–4.5-fold higher selectivity for KRAS over BRAF mutant 3D spheroid growth than OphA, suggesting improved relative on-target activity. Importantly, Calmirasone1 has a 40–260-fold lower unspecific toxic effect on HRAS mutant cells, while it reaches almost 50% of the activity of novel K-RasG12C specific inhibitors in 3D spheroid assays. Our results suggest that Calmirasone1 can serve as a new tool compound to further investigate the cancer cell biology of the K-Ras and CaM associated stemness activities.

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Section: Methodsmentioning

confidence: 99%

A Covalent Calmodulin Inhibitor as a Tool to Study Cellular Mechanisms of K-Ras-Driven Stemness

Okutachi

Manoharan

Kiriazis

et al. 2021

Front. Cell Dev. Biol.

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“…The assay utilizes ARC-Lum(Fluo) probe ARC-1063, which emits long lifetime photoluminescence signal when associated with a kinase target. 39,40 Hence, the Scheme 1. Synthesis of Acetal-Protected FMK Warhead (19) integrated time-gated luminescence intensity (TGLI) measured between 50 and 250 μs after flash excitation at 337 nm correlates with the concentration of ARC-Lum(Fluo)-PK complex in the solution.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Construction of Covalent Bisubstrate Inhibitor of Protein Kinase Reacting with Cysteine Residue at Substrate-Binding Site

et al. 2022

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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 k inact/K I 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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“…The IC 50 value was derived by fitting the log concentration of inhibitor vs fluorescence anisotropy signal in Prism (GraphPad) software. The IC 50 value was converted into K d as described previously [ 45 ].…”

Section: Methodsmentioning

confidence: 99%

Potential of phenothiazines to synergistically block calmodulin and reactivate PP2A in cancer cells

Manoharan

Okutachi²,

Abankwa³

2022

PLoS ONE

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Phenothiazines (PTZ) were developed as inhibitors of monoamine neurotransmitter receptors, notably dopamine receptors. Because of this activity they have been used for decades as antipsychotic drugs. In addition, they possess significant anti-cancer properties and several attempts for their repurposing were made. However, their incompletely understood polypharmacology is challenging. Here we examined the potential of the PTZ fluphenazine (Flu) and its mustard derivative (Flu-M) to synergistically act on two cancer associated targets, calmodulin (CaM) and the tumor suppressor protein phosphatase 2A (PP2A). Both proteins are known to modulate the Ras- and MAPK-pathway, cell viability and features of cancer cell stemness. Consistently, we show that the combination of a CaM inhibitor and the PP2A activator DT-061 synergistically inhibited the 3D-spheroid formation of MDA-MB-231 (K-Ras-G13D), NCI-H358 (K-Ras-G12C) and A375 (B-raf-V600E) cancer cells, and increased apoptosis in MDA-MB-231. We reasoned that these activities remain combined in PTZ, which were the starting point for PP2A activator development, while several PTZ are known CaM inhibitors. We show that both Flu and Flu-M retained CaM inhibitory activity in vitro and in cells, with a higher potency of the mustard derivative in cells. In line with the CaM dependence of Ras plasma membrane organization, the mustard derivative potently reduced the functional membrane organization of oncogenic Ras, while DT-061 had a negligible effect. Like DT-061, both PTZ potently decreased c-MYC levels, a hallmark of PP2A activation. Benchmarking against the KRAS-G12C specific inhibitor AMG-510 in MIA PaCa-2 cells revealed a higher potency of Flu-M than combinations of DT-061 and a CaM inhibitor on MAPK-output and a strong effect on cell proliferation. While our study is limited, our results suggest that improved PTZ derivatives that retain both, their CaM inhibitory and PP2A activating properties, but have lost their neurological side-effects, may be interesting to pursue further as anti-cancer agents.

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Binding assay for characterization of protein kinase inhibitors possessing sub-picomolar to sub-millimolar affinity

Cited by 16 publications

References 42 publications

A Covalent Calmodulin Inhibitor as a Tool to Study Cellular Mechanisms of K-Ras-Driven Stemness

A Covalent Calmodulin Inhibitor as a Tool to Study Cellular Mechanisms of K-Ras-Driven Stemness

Construction of Covalent Bisubstrate Inhibitor of Protein Kinase Reacting with Cysteine Residue at Substrate-Binding Site

Potential of phenothiazines to synergistically block calmodulin and reactivate PP2A in cancer cells

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