It should be noted that the long lifetime binding
It should be noted that the long-lifetime binding-responsive luminescence of ARC-1528 and ARC-1530 becomes evident only upon illumination with radiation at near-UV range where the excitation of ATB or ASB moiety is possible. At higher wavelength range (visible light), the fluorophore (5-TAMRA) can be directly excited and the compounds can be used like common fluorescent probes. The latter option extends the applicability of the probes to assays based on the measurement of fluorescence intensity and fluorescence polarization. The presented probes are not cell-permeable because of the large negative charge on the peptide moiety. However, we have recently shown that esterification of the peptoid moieties of similar compounds results in prodrugs with intracellular activity.19, 31 Therefore these luminescence probes could be converted into cell permeable compounds by using the previously described prodrug strategy.
Materials and methods Chemicals were purchased from ABCR, Acros, Alfa Aesar, Deutero GmbH, Iris Biotech GmbH, Fluka, Macherey-Nagel, Fisher Scientific, Sigma-Aldrich and TCI Chemicals. CX-4945 was from Synkinase. CK2α1-335 and CK2α’ proteins were kind gifts from Prof. O.-G. Issinger (University of Southern Denmark). THF was dried over molecular sieves. t-BuOK was purified by dissolving the crude material in THF, centrifugation and CGRP (rat) and of the supernatant. HPLC-MS was performed with Schimadzu LC Solution (Prominence) system connected to LCMS-2020 ESI-MS. Purification of the samples was performed with RP-HPLC equipped with a Phenomenex Luna C18 analytical column (5 μm, 250 × 4.6 mm) at 40 °C and photodiode array detector SPD-M20A. Acetonitrile/water + 0.1% aqueous TFA gradient at a flow rate of 1 mL/min was used. ESI-HRMS of compounds were measured with Thermo Electron LTQ Orbitrap spectrometer in positive ion mode. The concentrations of stock solutions of ligands and probes were determined spectrophotometrically (NanoDrop 2000c, Thermo Scientific) in the same buffer used in biochemical measurements. The following molar extinction coefficients were used: ATB-conjugates: ε322nm = 23 000 M−1 cm−1, ASB-conjugates: ε330nm = 21 000 M−1 cm−1 and 5-TAMRA-labeled conjugates: ε558nm = 80 000 M−1cm−1.
Acknowledgement This work was funded by a grant from the Estonian Research Council (IUT20-17).
Introduction In response to changes in environmental conditions, cells rapidly adjust gene expression programs to maintain cellular homeostasis and cell survival . In Saccharomyces cerevisiae, various types of stresses, such as heat shock, oxidative or reductive stress, osmotic shock, nutrient starvation, DNA damage, and extreme pH, lead to an extensive alternation of gene expression, termed the environmental stress response (ESR) , . The ESR genes are categorized into two groups based on their opposite expression patterns, which are stress-repressed genes and stress-induced genes . The stress-repressed ESR genes are mainly involved in protein synthesis, whereas stress-induced ESR genes are involved in a wide range of cellular processes to rapidly adapt to abrupt environmental changes. Expression of stress-induced ESR genes is primarily regulated by functionally redundant transcription factors Msn2 and Msn4, known as general stress transcription factors . The msn2Δmsn4Δ cells are hypersensitive to stressful conditions, indicating that Msn2/4 activity and expression levels of their target genes are crucial for yeast cell survival under various environmental challenges . Msn2/4 activate expression of a large number of genes containing stress response element (STRE) in their promoters in response to a variety of stresses, such as nutrient starvation, heat shock, osmotic shock, oxidative stress, alteration of pH, and noxious chemicals , , . Transcriptional activity of Msn2/4 is regulated at multiple levels, including nuclear translocation, DNA binding, and stability, which are primarily influenced by the phosphorylation status of Msn2/4 , , .