Structure‐based cross‐linking of NF‐κB p50 homodimer and decoy bearing a novel 2'‐disulfide trapping site

Metelev, Valeri; Romanenkov, A. S.; Кубарева, Е. А.; Zubin, Evgeniy; Polouchine, Nikolai; Zatsepin, Timofei S.; Molochkov, Nikolai V.; Oretskaya, Tatiana S.

doi:10.1080/15216540600981750

Cited by 14 publications

(4 citation statements)

References 15 publications

(14 reference statements)

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“…α, β-unsaturated ketones are known as Michael acceptors that potentially capture well-positioned nucleophiles at its target binding site, and may react with sulfhydryl groups of cysteine residues of proteins [ 27 , 28 ]. Human p50 has seven cysteine residues at positions 62, 88, 119, 124, 162, 262, and 273 [ 29 , 30 ]. It is known that residues 59-71 (RYVCEGPSHGGLP) of p50 constitute the DNA binding domain of NF-κB [ 31 – 34 ], among which the sulphydryl group of Cys62 is an important determinant of DNA recognition by p50 subunit of NF-κB [ 35 ].…”

Section: Resultsmentioning

confidence: 99%

Identification and validation of p50 as the cellular target of eriocalyxin B

et al. 2014

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As an ent-kaurene diterpenoid isolated from Isodon eriocalyx var. Laxiflora, Eriocalyxin B (EriB) possesses potent bioactivity of antitumor and anti-autoimmune inflammation, which has been suggested to work through inhibition of NF-kappaB (NF-κB) signaling. However, the direct target of EriB remains elusive. In this study, we showed that EriB induced apoptosis is associated with the inhibition of NF-κB signaling in SMMC-7721 hepatocellular carcinoma cells. With activity-based probe profiling, we identified p50 protein as the direct target of EriB. We showed that cysteine 62 is the critical residue of p50 for EriB binding through the α, β-unsaturated ketones. As the result, EriB selectively blocks the binding between p50 and the response elements, whereas having no effect on the dimerization or the nuclear translocation of p50 and p65. SiRNA mediated knockdown of p50 attenuated the apoptosis induced by EriB in SMMC-7721 cells. Taken together, our studies illustrated that EriB induces cancer cell apoptosis through interfering with the binding between NF-κB and the response elements by targeting the cysteine 62 of p50, which highlights its potential for the development of p50 targeted cancer therapeutic agents.

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Section: Resultsmentioning

confidence: 99%

Identification and validation of p50 as the cellular target of eriocalyxin B

et al. 2014

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“…We show for the first time that the Cys 62 residue in the active site of NF-nB (p50) protein was modified by BSC through the formation of a -Se-S-bond. The thiol group of Cys 62 is critical in the DNA recognition by p50 subunit (31,(41)(42)(43)(44) and is the only cysteine, among the seven cysteines present in p50, located in the DNA recognition region (44). The formation of -Se-S-bond may be due to an S N 2 reaction by a nucleophilic attack of the thiol group of Cys 62 at the electrophilic selenium moiety of the selenocyanate with displacement of the cyano group; however, an S N 1 reaction mechanism cannot be excluded.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Nuclear Factor-κB DNA Binding by Organoselenocyanates through Covalent Modification of the p50 Subunit

Chen¹,

Spratt²,

Stanley³

et al. 2007

Cancer Research

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Most known chemopreventive agents including certain selenium compounds suppress the activation of the nuclear factor KB (NF-KB), but the mechanisms remain largely elusive. Toward this end, we initially showed that the inhibition of NF-KB DNA binding by benzyl selenocyanate (BSC) and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) was reversed by the addition of DTT; this suggests the formation of DTT-reducible selenium-sulfur bonds between selenocyanate moieties and cysteine residues in NF-KB (p50) protein.Furthermore, the inhibitory effect of selenocyanates on NF-KB was not altered in the presence of physiologic level of reduced glutathione (1 mmol/L), suggesting that selenocyanates can also inhibit NF-KB in vivo. Using both matrixassisted laser desorption/ionization-time of flight and tandem mass spectrometry fragmentation, we showed for the first time that the Cys 62 residue in the active site of NF-KB (p50) protein was modified by BSC through the formation of a selenium-sulfur bond. In addition, p-XSC-bound NF-KB (p50) protein was also detected by a radiotracer method. To provide further support, molecular models of both BSC and p-XSC positioned in the DNA binding pocket of the p50 were constructed through the covalent modification of Cys 62 ; the models reveal that DNA substrate could be hindered to enter its DNA binding region. This study shows for the first time that BSC and p-XSC may exert their chemopreventive activity, at least in part, by inhibiting NF-KB through covalent modification of Cys 62 of the p50 subunit of NF-KB. [Cancer Res 2007;67(21):10475-83]

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“…This thiol-disulfide exchange is rapid and irreversible because the pyridinesulfenyl group is a good leaving group. 22,23 The thiolate anion was generated under basic conditions from the deprotection of a 2′-O-acetylthiomethyl (2′-O-AcSM) derivative previously studied in our lab (Scheme 1). 8 In the partially modified 2′-O-RSSM/2′-OH RNAs, the 2′-OH resulted from the removal of 2′-O-pivaloyloxymethyl (2′-O-PivOM) groups under the standard ammonia treatment.…”

Section: Synthesis Of 2′-o-rssm Oligonucleotidesmentioning

confidence: 99%

A versatile post-synthetic method on a solid support for the synthesis of RNA containing reduction-responsive modifications

et al. 2016

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An original post-synthetic method on a solid support was developed to introduce various disulfide bond containing groups at the 2'-OH of oligoribonucleotides (RNAs). It is based on a thiol disulfide exchange reaction between several readily accessible alkyldisulfanyl-pyridine derivatives and 2'-O-acetylthiomethyl RNA in the presence of butylamine. By this strategy, diverse 2'-O-alkyldithiomethyl RNAs were obtained. These modifications provided high nuclease resistance to RNA and were easily removed with glutathione treatment, thus featuring a potential use for siRNA prodrugs.

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Structure‐based cross‐linking of NF‐κB p50 homodimer and decoy bearing a novel 2'‐disulfide trapping site

Cited by 14 publications

References 15 publications

Identification and validation of p50 as the cellular target of eriocalyxin B

Identification and validation of p50 as the cellular target of eriocalyxin B

Inhibition of Nuclear Factor-κB DNA Binding by Organoselenocyanates through Covalent Modification of the p50 Subunit

A versatile post-synthetic method on a solid support for the synthesis of RNA containing reduction-responsive modifications

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