Cyclic 5-membered disulfides are not selective substrates of thioredoxin reductase, but are opened nonspecifically

The cyclic five-membered disulfide 1,2-dithiolane has been widely used in chemical biology and in redox probes. Contradictory reports have described it either as nonspecifically reduced in cells, or else as a highly specific substrate for thioredoxin reductase (TrxR). Here we show that 1,2-dithiolane probes, such as “TRFS” probes, are nonspecifically reduced by thiol reductants and redox-active proteins, and their cellular performance is barely affected by TrxR inhibition or knockout. Therefore, results of cellular imaging or inhibitor screening using 1,2-dithiolanes should not be interpreted as reflecting TrxR activity, and previous studies may need re-evaluation. To understand 1,2-dithiolanes’ complex behaviour, probe localisation, environment-dependent fluorescence, reduction-independent ring-opening polymerisation, and thiol-dependent cellular uptake must all be considered; particular caution is needed when co-applying thiophilic inhibitors. We present a general approach controlling against assay misinterpretation with reducible probes, to ensure future TrxR-targeted designs are robustly evaluated for selectivity, and to better orient future research

Identification of novel small molecules that inhibit STAT3-dependent transcription and function

Activation of Signal Transducer and Activator of Transcription 3 (STAT3) has been linked to several processes that are critical for oncogenic transformation, cancer progression, cancer cell proliferation, survival, drug resistance and metastasis. Inhibition of STAT3 signaling has shown a striking ability to inhibit cancer cell growth and therefore, STAT3 has become a promising target for anti-cancer drug development. The aim of this study was to identify novel inhibitors of STAT-dependent gene transcription. A cellular reporter-based system for monitoring STAT3 transcriptional activity was developed which was suitable for high-throughput screening (Z’ = 0,8). This system was used to screen a library of 28,000 compounds (the ENAMINE Drug-Like Diversity Set). Following counter-screenings and toxicity studies, we identified four hit compounds that were subjected to detailed biological characterization. Of the four hits, KI16 stood out as the most promising compound, inhibiting STAT3 phosphorylation and transcriptional activity in response to IL6 stimulation. In silico docking studies showed that KI16 had favorable interactions with the STAT3 SH2 domain, however, no inhibitory activity could be observed in the STAT3 fluorescence polarization assay. KI16 inhibited cell viability preferentially in STAT3-dependent cell lines. Taken together, using a targeted, cell-based approach, novel inhibitors of STAT-driven transcriptional activity were discovered which are interesting leads to pursue further for the development of anti-cancer therapeutic agents

Selective, Modular Probes for Thioredoxins Enabled by Rational Tuning of a Unique Disulfide Structure Motif

Specialised cellular networks of oxidoreductases coordinate the dithiol/disulfide-exchange reactions that control metabolism, protein regulation, and redox homeostasis. For probes to be selective for redox enzymes and effector proteins (nM to µM concentrations), they must also be able to resist nonspecific triggering by the ca. 50 mM background of non-catalytic cellular monothiols. However, no such selective reduction-sensing systems have yet been established. Here, we used rational structural design to independently vary thermodynamic and kinetic aspects of disulfide stability, creating a series of unusual disulfide reduction trigger units designed for stability to monothiols. We integrated the motifs into modular series of fluorogenic probes that release and activate an arbitrary chemical cargo upon reduction, and compared their performance to that of the literature-known disulfides. The probes were comprehensively screened for biological stability and selectivity against a range of redox effector proteins and enzymes. This design process delivered the first disulfide probes with excellent stability to monothiols, yet high selectivity for the key redox-active protein effector, thioredoxin. We anticipate that further applications of these novel disulfide triggers will deliver unique probes targeting cellular thioredoxins. We also anticipate that further tuning following this design paradigm will deliver redox probes for other important dithiol-manifold redox proteins, that will be useful in revealing the hitherto hidden dynamics of endogenous cellular redox systems.</p

Cyclic 5-Membered Disulfides Are Not Selective Substrates of Thioredoxin Reductase, but Are Opened Nonspecifically

The cyclic five-membered disulfide 1,2-dithiolane has been used as the key element in numerous chemical biology probes. Contradictory views of this disulfide populate the literature: some reports describe it as being nonspecifically reduced, others as a highly specific substrate for thioredoxin reductase (TrxR). We here show that 1,2-dithiolane probes are nonspecifically reduced by a broad range of thiol reductants and redox-active proteins, and that their cellular performance is barely affected by TrxR inhibition or knockout. We conclude that inhibitor screenings and "TRFS" probes that have used 1,2-dithiolanes as TrxRselective substrates should be treated with caution, and may need re-evaluation. Understanding 1,2-dithiolanes’ behaviour needs consideration of probe localisation and environmentdependent fluorescence, reduction-independent ring-opening polymerisation, thiol-dependent cellular uptake, and caution when applying thiophilic inhibitors. We present an approach controlling against assay misinterpretation with reducible probes, to ensure that future TrxR-targeted designs are robustly evaluated for selectivity, and to better orient future research

Characterization of the four hit compounds.

<p><b>(A-D)</b> Compounds KI1, KI4, KI12 and KI16 and their corresponding activities in the luciferase reporter assays (center) and cell viability assays (right). Luciferase assays were performed in A4wt cells stimulated with IL6 (grey) and in A4 cells stimulated with IFNγ (red). Viability assays were conducted over 48 hours in A4wt cells (grey) and A4 cells (red). <b>(E)</b> Luciferase IC₅₀ values for lead compounds. Curves were fit using GraphPad and error bars indicate the 95% confidence intervals. <b>(F)</b> Cell viability EC₅₀ values in A4 and A4wt cells using CellTiterGlo assay (Promega Biotech AB, Sweden). Curves were fit using GraphPad and error bars indicate the 95% confidence intervals.</p

Primers used for qRT-PCR.

<p>Primers used for qRT-PCR.</p

Docking assays and chemical features of the lead compounds.

<p><b>(A)</b> Chemical structures of KI1 and KI4. The quinazolone core of KI1 and KI4 is also a common feature of clinically approved EGFR- and VEGFR-targeted therapeutics Gefitinib, Erlotinib and Vandetanib. The pharmacophore portion of the clinically used compounds is highlighted in blue, which is highly similar to the structures of KI1 and KI4. <b>(B)</b> STAT3 crystal structure (blue, pdb 1BG1) with residues 702–709 of the opposing monomer shown in cyan. <b>(C)</b> (Upper panel) KI12 docked into the STAT3 SH2 domain using GLIDE docking software. (Lower panel) Ligand interaction diagram showing interactions between KI12 and residues on the STAT3 SH2 domain. KI12 hydrogen bonds with Ser611, Ser613 and Glu612 and forms cation-pi type interactions with the positively charged Lys591. An additional hydrogen bond is formed with Gln635 and a salt bridge with Lys626. <b>(D)</b> (Upper panel) KI16 docked into the STAT3 SH2 domain using GLIDE docking software. (Lower panel) Ligand interaction diagram showing interactions between KI16 and residues on the STAT3 SH2 domain. KI16 makes hydrogen bonds with Ser611, Ser613 and Gln635.</p

The effect of the lead compounds on the IL6-JAK-STAT pathway.

<p><b>(A)</b> A4wt or A4 cell lines were pre-treated with the indicated compounds (5 and 10μM) for 30 min followed by stimulation with IL6+sIL6R (50 and 100 ng/mL, respectively) for 30 min. Levels of phosphorylated STAT3 (pTyr705) and STAT1 (pTyr701) and total levels of these proteins were determined by Western blotting analysis. GAPDH was used as a loading control. Both left and right panels of western blots were performed simultaneously. <b>(B)</b> A4wt and A4 cell lines were pre-treated with the indicated compounds (5 and 10μM) for 30 min followed by stimulation with IFNγ (40 IU/mL) for 30 min. Levels of phospho-STAT1 and total STAT1 were determined by Western blotting analysis. GAPDH was used as a loading control. <b>(C)</b> A4wt cells were pre-treated with the compounds KI1, KI4, KI12 and KI16 in the concentration of 10 μM and then treated with IL6 + sIL6R (50 ng/mL and 100 ng/mL respectively) for 30 min. The cell pellets were lysed and pJAK1, pJAK2 and total JAK1 and JAK2 levels were assessed by Western blotting. GAPDH was used as a loading control. <b>(D)</b> A4wt cells were stimulated with either IL6+sIL6R (50 and 100 ng/mL, respectively) or IFNγ (40 IU/mL) for 4 h. Expression of indicated STAT3 target genes was assessed by qRT-PCR. The data represents mean of duplicates ± SD. <b>(E)</b> A4wt cells were pretreated with the indicated compounds (5 and 10 μM) for 30 min followed by IL6+sIL6R stimulation for 4 h (as in <b>D</b>). The expression of indicated STAT3 target genes was determined by qRT-PCR. The data is normalized to β-actin expression and presented as fold expression relative to the untreated control. The data represents mean of duplicates ± SD.</p

Differential effect of the lead compounds on viability of STAT3-dependent and STAT3-independent cancer cell lines.

<p><b>(A)</b> Cell pellets from the prostate cancer cells lines PC3 and DU145, and the breast cancer cell lines MCF7 and MDA-MB468, were lysed and subjected to Western blotting analysis. Antibodies against pSerSTAT3, pTyrSTAT3, pTyrSTAT1 and the corresponding antibodies for the total levels of these proteins were used. GAPDH was used as a loading control. <b>(B)</b> PC3, DU145, MCF7 and MDA-MB468 cells were seeded in 384-well white plates using Multidrop. Cells were treated with the range of compounds concentration using Echo liquid dispenser. Cell viability was assessed after 48h using CellTiterGlo viability assay (Promega) according to the instructions of the manufacturer. The viability of the DMSO-treated cells was set as 100%. The data represents IC50 values for the four cell lines calculated from three replicates ± SD. <b>(C, D)</b> DU145 <b>(C)</b> and MDA-MB468 <b>(D)</b> cells were treated with the indicated compounds for 4 h. Levels of phospho-STAT3 and total STAT3 were determined by Western blotting analysis. GAPDH was used as a loading control. <b>(E)</b> DU145 cell line was transfected with 50 nM scrambled siRNA or STAT3 siRNA for 48 h or treated with the indicated compounds for 16 h and 24h, respectively. The concentrations of the compounds were taken from the IC₅₀ values for viability (<b>3B</b>). Expression of the indicated STAT3 target genes was analysed by qRT-PCR. The values are normalized to the expression of GUSB and presented as a relative expression to untreated controls. Error bars represent mean + SE from three independent experiments. <b>(F</b>) A wound healing assay was performed using DU145 cells treated with the indicated compounds at 20 μM. Migration into the denuded area was monitored after 4, 8 and 24 h. The data represents mean of triplicates ± SE from three independent experiments. * p < 0.05.</p

Cellular system for screening of compounds inhibiting STAT3 transcriptional activity.

<p><b>(A)</b> Overview of the STAT3 luciferase reporter system. <b>(B)</b> A4wt and A4 sublines were transiently transfected with pGL4.27-SIE reporter and pRL-Renilla and either left untreated or were treated with IL6 + IL6R (50ng/mL and 100 ng/mL respectively) or IFNγ (40 IU/mL). After 6h, luciferase activities were measured using Dual-Glo Luciferase assay. The data presented as ratios to the corresponding untreated control. <b>(C)</b> A4 and A4wt sublines were treated with IL6 + sIL6R (50ng/mL and 100 ng/mL, respectively) or IFNγ (40 IU/mL) for 30 min. Western blotting analysis was performed with the indicated antibodies. GAPDH was used as a loading control. <b>(D)</b> A4wt cells were transfected with pGL4.27-SIE reporter and a stable sub-line A4wt-SIE-6 was selected. Cells were either left untreated (control) or were stimulated with IL6 + sIL6R (50 and 100 ng/mL, respectively) either before treatment with STATTIC (10 μM) or Pyr6 (1 μM), or after, as indicated. Luciferase activity was measured 6h after the first treatment in both cases using SteadyLite^TM Plus. The data is presented as fold induction. Error bars represent SE from quadruplicates. <b>(E)</b> A4wt cells stably transfected with pGL4.27-SIE reporter were seeded in 384-well plates and pre-treated with IL6 and sIL6R (50 and 100 ng/mL, respectively) for 1h, whereafter treated with STATTIC (10 μM) or Pyr6 (1 μM) for 5h to estimate the suitability of the screening system for the high-throughput screening and calculate the z’-factor. Luciferase activity was measured as in (C). One column of the 384-well plate was used for each treatment. The data is presented as average of raw luciferase activity ± standard deviation. The relevant z’-values are described in the text. <b>(F)</b> Outline of the luciferase reporter assay used for high-throughput screening. <b>(G)</b> The dot-blot of the results of the primary screening. A4wt-SIE-6 cells stably transfected with pGL4.27-SIE were pre-treated with IL6 and sIL6R and then treated with the drugs. The data are presented as percent inhibition of luciferase activity where the luciferase activity of the cells treated with IL6+sIL6R+mock is regarded as 0% inhibition (red dots), and the activity in cells treated with IL6+sIL6R+STATTIC regarded as 100% inhibition (green dots). The dashed line is the distance of 3 standard deviations from IL6+sIL6R+mock-treated cells. All the library compounds were used at 10 μM. <b>(H</b>) The STAT3 inhibitor screening funnel.</p