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residue proline at the position 123 of the hinge region, where luminescence signal of the probes with the PK Pim-1 points to
other PKs have a valine residue. The proline residue lacks the the possibility that binding of a benzoselenadiazole fragment
hydrogen bond donor ability for binding ligands.15 This bond to the ligand-binding pocket of a protein may be not sufficient
of the luminescent donor seems to be important for formation for the stabilization of the triplet state of the phosphor. A specific
or stabilization of the triplet state of the latter. CK2 with Val116 interaction of the protein with the benzoselenadiazole fragment
is able to give the required hydrogen bond. Indeed, binding of is needed to fix the donor luminophore in a suitable conforma-
ARC-3141 with CK2 in addition to the increase of the fluorescence tion for productive electron transfer and prevent access of
anisotropy value of the PF647 dye [lex = 590 nm, lem = 675 nm] oxygen and other quenchers. Thus the development of respon-
also led to a strong luminescence signal [lex = 337 nm, lem
=
sive probes for some of benzoselenadiazole-binding proteins
675 nm] with a lifetime of 20 Æ 2 ms (Fig. 1D and Fig. S4, ESI†). could not be achieved. On the other hand, the obtained knowl-
Thereat the binding of the parent conjugate comprising no edge confirms the previous understanding that non-specific
fluorescent dye (ARC-3138) with CK2 revealed a weak phos- associations of ARC-Lum(Fluo)-probes with proteins, other bio-
phorescence emission signal with a long decay time in the polymers and small molecules do not result in a microsecond-
complex with CK2, but the intensity of this signal [lex = 337 nm, scale photoluminescence.
lem = 675 nm] was about 400-fold lower than the signal from
This work was supported by grants from the Estonian
the ARC-3141–CK2 complex (Fig. S5, ESI†). Interestingly, ARC- Ministry of Education and Sciences (SF0180121s08) and the
3141 showed a higher affinity towards CK2 (KD = 25 Æ 9 nM) Estonian Science Foundation (8230, 8419 and ERMOS30) and
than ARC-3138 (KD = 82 Æ 22 nM), revealed both by binding/ the Estonian Research Council (IUT20-17). SK is grateful for
displacement and inhibition assays (Fig. 1, Fig. S6 and S7, financial support from the SGC, a registered charity (number
ESI†). This is apparently caused by masking the unfavourable 1097737) that receives funds from the Canadian Institutes
effect of the positive charge of lysine in the C-terminus of the for Health Research, the Canada Foundation for Innovation,
conjugate, as the acetylation of the amino group increased the Genome Canada, GlaxoSmithKline, Pfizer, Eli Lilly, the Novartis
affinity in the same way (Fig. S7, ESI†). The high affinity of ARC- Research Foundation, Takeda, the Ontario Ministry of Research
3141 to CK2 enables the application of the probe for measure- and Innovation and the Wellcome Trust. OGI is grateful for
ment of the concentration of CK2 and characterization of CK2 support from the Danish Cancer Society (DP07109). The
inhibitors both in buffer solutions and cell lysates. Selectivity authors thank Gerda Raidaru and Tine D. Rasmussen for
profiling of ARC-3138 in the panel of 140 protein kinases excellent technical help.
revealed its clear CK2 selectivity (Table S3, ESI†). At 1 mM
concentration it inhibited 80% of CK2 activity while all other
kinases were inhibited by less than 50%.
In conclusion, the work demonstrates that benzoselena-
diazole is a promising novel scaffold that possesses the property
Notes and references
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4098 | Chem. Commun., 2014, 50, 4096--4098
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