Calmodulin is labeled at lysine 148 by a chemically reactive phenothiazine.

Faust, Frederick M.; Slisz, M.; Jarrett, Harry W.

doi:10.1016/s0021-9258(18)61599-6

Cited by 35 publications

(24 citation statements)

References 22 publications

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“…Differential kinetic labelling experiments suggested that binding of trifluoperazine to Ca 2+ /CaM perturbs lysines 75, 77 and 148, the same residues that are modified by the covalent CaM inhibitor ophiobolin A [ 7 ]. In agreement with the kinetic labelling studies, chemically reactive PTZ modified the same lysines [ 8 ]. Efforts were made to generate covalently binding derivatives of this compound class, as irreversible binding could significantly increase the potency of drugs [ 9 ].…”

Section: Introductionsupporting

confidence: 82%

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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Section: Introductionsupporting

confidence: 82%

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

Manoharan

Okutachi²,

Abankwa³

2022

PLoS ONE

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“…Aliquots of the photolysis mixtures containing 10 fig of modified and native calmodulins were mixed with 40 mL of packed streptavidin-agarose (extensively prewashed with 100 mM NaH2P04 at pH 7.0 followed by 100 mM HEPES at pH 7.4). Parallel analyses were performed on control samples containing 10 order to correct for recovery and dilution during processing. After incubation for 1 h at 25 °C with agitation on a rotary shaker, the supernatant fractions containing unbound CaM were harvested following centrifugation in a microfuge (30 s).…”

Section: Methodsmentioning

confidence: 99%

“…Previous studies of CaM using phenothiazines possessing a chemically reactive moiety incorporated in a side chain (8)(9)(10) yielded modification of Lys residues adjacent to the Nand/or C-terminal hydrophobic pockets. In each case, however, modification of CaM occurred outside the hydrophobic binding pockets and required detailed studies to determine the specific site of modification.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and Use of a Biotinylated 3-Azidophenothiazine to Photolabel Both Amino- and Carboxyl-Terminal Sites in Calmodulin

Delaluz¹,

Golinski²,

Watt³

et al. 1995

Bioconjugate Chem.

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The biotinylated probe, 3-azido-10-(4-(4-biotinyl-1-piperazinyl)butyl)phenothiazine, was used to examine the phenothiazine binding domains in calmodulin (CaM) by photolabeling. This phenothiazine, synthesized from 3-azido-10-(4-(1-piperazinyl)butyl)phenothiazine and d-biotinyl tosylate, inhibited the CaM-mediated activation of phosphodiesterase (PDE) with an I50 of 12.5 (+/- 2.8) microM. Photolabeling of CaM produced covalent adducts in excellent yield (32%) in a light- and Ca2+-dependent manner. Studies performed over a range of drug concentrations suggested a 2:1 stoichiometry for the binding of the phenothiazine probe to CaM. Limited trypsin digestion and purification of the resulting fragments by either SDS-PAGE or HPLC provided two principal phenothiazine-containing peptides. Amino acid composition and sequence analyses performed on these two peptides established that both the N- and C-terminal domains in CaM, particularly the regions amino terminal to Ca2+-binding loops 1 and 3, were modified by the photoactivated phenothiazine derivative. These data, particularly for the C-terminal domain, are in excellent agreement with the X-ray structure analysis of a 1:1 CaM-trifluoperazine complex.

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“…It forms an irreversible covalent adduct via C5, C21-dicarbonyl functionalities after intermediate Schiff base formation with Lys 75 or Lys 77 and Lys 148 of CaM ( Supplementary Scheme 1 ). Thus, OphA can react with CaM at a 2:1 ratio, similar to covalent phenothiazine derivatives, which also react with the same lysines ( Faust et al, 1987 ). Despite its potency against CaM, OphA appears to have several other targets, such as phosphatidylethanolamine ( Chidley et al, 2016 ).…”

Section: Introductionmentioning

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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Calmodulin is labeled at lysine 148 by a chemically reactive phenothiazine.

Cited by 35 publications

References 22 publications

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

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

Synthesis and Use of a Biotinylated 3-Azidophenothiazine to Photolabel Both Amino- and Carboxyl-Terminal Sites in Calmodulin

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

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