Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, Intact protein LC–MS spectra of Pin1 (black) directly identify covalent binders (blue) in the electrophilic library screen (200 μM compound for 24 h). Madduct indicates the mass of the expected adduct for the indicated example. b, Distribution of hits in the Pin1 screening campaign and their corresponding labeling (%). Nine hits (18.75%) out of the 48 top hits that labeled Pin1 at >75% (dark and light blue) share sulfolene or sulfolane moieties. Labeling percentage calculated as previously described28. c, 2D analysis of the top ten optimized binders (structures shown in f); labeling percentage in the LC–MS assay plotted against reactivity (log (K)) suggests Sulfopin for further biological evaluation. d, Fluorescence polarization assay with the top ten binders, including juglone and a nonreactive control (Sulfopin-AcA), after 14 h of preincubation with Pin1. Data points are plotted as the average of n = 3 independent samples ± s.e.m., and are representative of n = 2 independent experiments. See Supplementary Table 3 for apparent Ki. mP represents the polarization value. e, PPIase substrate activity assay of Pin1 with Sulfopin (n = 3) and juglone (n = 2). Data points are plotted as the average of independent experiments ± s.e.m. for Sulfopin. f, Structures of the top ten binders in the Pin1-labeling LC–MS assay, the nonreactive control Sulfopin-AcA and juglone. g, X-ray crystal structure of Pin1 in complex with Sulfopin (1.4-Å resolution, PDB code 6VAJ). Pin1 (white) with relevant side chains in stick representation; Sulfopin is shown in pink. Hydrogen bonds are depicted as dashed lines. AU, arbitrary units.

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Liquid Chromatography with Mass Spectroscopy, Labeling, Fluorescence, Activity Assay

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, Fluorescence polarization assay showing that the DTB-labeled probe, Sulfopin-DTB, binds Pin1 with similar potency to Sulfopin following 14 h of incubation with Pin1. Data points are plotted as the average of n = 3 independent samples ± s.e.m., and are representative of n = 2 independent experiments. b, Chemical structure of Sulfopin-DTB. c, Sulfopin shows time-dependent engagement in PATU-8988T cells. PATU-8988T cells were treated with Sulfopin (1 μM) for the indicated time points followed by cell lysis, incubation with Sulfopin-DTB (1 μM), streptavidin pulldown and immunoblot analysis. d,e, Sulfopin shows long-term engagement of Pin1. PATU-8988T (d) or HCT116 (e) cells were incubated with or without Sulfopin for the indicated time points, followed by cell lysis, incubation with DTB probe, streptavidin pulldown and immunoblot analysis. Substantial engagement (>50%) was still evident after 72 h. f,g, Sulfopin fully engages Pin1 in PATU-8988T cells at 1 μM and in HCT116 cells at 0.5 μM (see Supplementary Fig. 9b for the structure of BJP-DTB). PATU-8988T (f) or HCT116 (g) cells were incubated with Sulfopin at the indicated concentrations for 5 h, followed by cell lysis, DTB probe incubation (1 h, 1 μM), streptavidin pulldown and immunoblot analysis. The noncovalent control, Sulfopin-AcA, is unable to outcompete Pin1 pulldown. c–g, Results are representative of n = 2 independent experiments. h, Sulfopin engages Pin1 in vivo. Mice were treated by oral gavage with the indicated amounts of Pin1 over 2 days for a total of three doses. Following this treatment, spleens were lysed for a competition pulldown experiment with Sulfopin-DTB. Results are representative of n = 2 independent pulldown experiments, starting from the same spleen lysates.

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Fluorescence, Labeling, Incubation, Lysis, Western Blot, In Vivo

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, CITe-Id profiling results showing Sulfopin-DTB-labeled cysteine sites, rank ordered by competitive dose response to Sulfopin. Out of 162 cysteine residues reproducibly labeled by Sulfopin-DTB in n = 2 independent experiments, Pin1 C113 was the only site identified with a competitive dose response >2 s.d. from the mean value of the null. (see Supplementary Dataset 3a for a full list of identified peptides, and Supplementary Fig. 10 for results with 12/24-h treatment). b, Waterfall plot showing competitive dose dependency of Pin1 C113 labeling in the CITe-Id experiment. Bars represent mean of n = 2 independent experiments. c, Out of 2,134 cysteines identified in the rdTOP-ABPP experiment, only two showed a light/heavy ratio of >2.5 and, of these, one did not replicate and only Pin1 C113 showed the maximal ratio of 15 in both replicates.

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Labeling

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, HeLa cells were treated with either DMSO, Sulfopin, or Go6976 (a Chk1 inhibitor) and exposed to 7.5 Gy IR 1 h after drug treatment. Viability was assessed 3 days post-IR. Sulfopin shows a dose dependent sensitization of the cells to irradiation (n=3; data are represented as mean values with standard deviation). b, Western blot analysis was performed 24 h post-IR, showing Sulfopin blocked phosphorylation of Thr209 of IRAK1. c, A shorter exposure shows that Sulfopin inhibits IRAK1 phosphorylation already at concentrations of 0.1 μM. d, A scheme for testing the effect of Sulfopin in vivo on germinal center B cells in response to immunization. e, Representative flow cytometric plots with Vehicle and Sulfopin (left) and quantification (right) of FASHi CD38− germinal center (GC) cells in WT mice 11 days after immunization with NP-OVA. ** p<0.01, two tailed Student’s t test.

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Knock-Out, Irradiation, Standard Deviation, Western Blot, In Vivo, Two Tailed Test

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, We previously27 generated a PATU-8988T Pin1 knockout (KO) cell line (Supplementary Fig. 12a). Sulfopin (1 μM) had a significant effect on cellular viability after 6 and 8 days (P = 0.01 and P = 0.01, respectively) in WT PATU-8988T cells (left), but showed no significant effect on viability in Pin1 KO cells (right); day 0-normalized growth rate for n = 3 biologically independent samples. b, Relative viability of PATU-8988T WT and Pin1 KO cells grown in 100% Matrigel domes following treatment with either Sulfopin (1 μM; n = 9 biologically independent samples; P = 1.24 × 10−18) or the noncovalent negative control, Sulfopin-AcA (1 μM; n = 9 biologically independent samples). Sulfopin-AcA showed no effect in any of the tested systems. c, Proportion of cells in various cell cycle stages as a function of Sulfopin treatment. The viability effects of Sulfopin are mediated by delayed cell cycle. PATU-8988T cells were treated with either DMSO, 2.5 μM Sulfopin or Sulfopin-AcA for 4 days. Cell cycle analysis was performed by BrdU and propidium iodide staining, followed by FACS analysis. Sulfopin treatment reduced the percentage of cells in S phase (P = 0.0004) and, in turn, increased the number of cells found in G1 phase (P = 0.003), while the noncovalent Sulfopin-AcA did not show this effect (n = 4; see Extended Data Fig. 3 for representative FACS analysis graphs and quantification of the results from two independent experiments). d, Cell culture growth curves. Sulfopin showed variation in antiproliferative effects across cancer cell lines Kuramochi, MDA-MB-468, NGP and NBL-S, with the most pronounced sensitivity observed in MDA-MB-468 cells (day 0-normalized growth rate for n = 3 biologically independent samples; P values for 2.5 μM Sulfopin after 4, 6 and 8 days were 0.007, 0.004 and 0.0004, respectively). Importantly we noted significant viability effects in Myc-high neuroblastoma cell lines NGP and NBL-S (P = 0.018 and 0.002, respectively for 2.5 μM Sulfopin after 8 days). Data points were plotted as the average of n = 3 biologically independent samples ± s.e.m. Statistical significance for all panels was calculated using one-tailed Student’s t-test with unequal variance (NS, not significant; P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Generated, Knock-Out, Negative Control, Cell Cycle Assay, Staining, Cell Culture, One-tailed Test

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, PATU-8988T cells were treated for 5 or 6 days with either DMSO (0.1%), Sulfopin (1 μM, 2.5 μM) or the non-covalent control Sulfopin-AcA (2.5 μM). The cells were lysed and activation of caspase 3 and Pin1 levels were analysed by Western blot. As a positive control for caspase 3 activation the cells were treated with Staurosporin (1 μM, 4h; STS). See Supplementary Fig. 13a for the results of an additional independent experiment. Caspase 3 was not activated and Pin1 levels were not changed by the treatment with Sulfopin. b, PATU-8988T cells were treated in triplicates for 6 days with either DMSO (0.1%), Sulfopin (1 M, 2.5 M) or the non-covalent control Sulfopin-AcA (2.5 M). The cells were then stained with AnnexinV-FITC/ 7AAD and analysed by FACS. Staurosporin treatment (1 M, 4h) was used as a positive control for apoptosis. Representative FACS analysis graphs and a quantification of the results (n=3; data are represented as mean values with standard deviation). See Supplementary Fig. 13b for the results of an additional independent experiment. Live cells were defined as AnnexinV−/7AAD−, early apoptosis AnnexinV+/7AAD− and late apoptosis AnnexinV+/7AAD+.

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Activation Assay, Western Blot, Positive Control, Staining, Standard Deviation

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, Results of an RNA-seq experiment comparing changes in RNA levels between Mino B cells treated with either Sulfopin (1 μM, 6 h, in triplicate) or DMSO. Each dot represents log2 fold change of a transcript (x axis) versus the P value for significance of that change (y axis; Wald test, as implemented in DESeq2). The dotted line indicates P = 0.05; 206 genes were significantly downregulated. b, Results of gene set enrichment analysis using Enrichr against the ENCODE TF chromatin immunoprecipitation–sequencing set. Two of the sets most enriched were Myc target genes from different cell lines. c, HEK293 cells were transfected with 4× E-box luciferase reporter for Myc transcriptional activity levels. Cotransfection with Pin1 increased reporter activity, while 48-h treatment with Sulfopin significantly (one-tailed Student’s t-test) reduced this activity compared to DMSO (n = 3; error bars indicate s.d.).

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: RNA Sequencing Assay, ChIP-sequencing, Transfection, Luciferase, Activity Assay, Cotransfection, One-tailed Test

Journal:

Article Title: Simultaneous De Novo Identification of Molecules in Chemical Mixtures by Doubly Indirect Covariance NMR Spectroscopy

doi: 10.1021/ja106781r

Figure Lengend Snippet: Doubly indirect covariance spectroscopy of cancer cell line DU145 extract. A) DQF-COSY spectrum, B) 13C-1H HSQC spectrum, C,D) doubly indirect 13C-13C covariance spectrum constructed from parent spectra A, B (see Eq. (2)) before (C) and after (D) removing effects due to 1H overlap, E) carbon-carbon connectivity graphs of mixture components determined from spectrum D. F) Labels a, b, c, d, e, f1 and f2 in Panels E, F indicate ethanol, lactate, taurine, myo-inositol, glutamate, and glutathione reduced, respectively. Graph g in panel E does not match any of the compounds in the database. Myo-inositol displays only 3 nodes (graph d in Panel E) because of 13C chemical shift degeneracy induced by its molecular symmetry.

Article Snippet: An extract from human prostate cancer cell line DU145 (from the American Type Culture Collection (ATCC), www.atcc.org ) was obtained as follows: 14 , 15 the cells were cultured at 37°C in 5% CO 2 incubator in DMEM medium supplemented with 10% fetal bovine serum and penicillin/streptomycin.

Techniques: Spectroscopy, Construct

Journal: Cell Death & Disease

Article Title: GNA13 regulates BCL2 expression and the sensitivity of GCB-DLBCL cells to BCL2 inhibitors in a palmitoylation-dependent manner

doi: 10.1038/s41419-020-03311-1

Figure Lengend Snippet: A The scheme of isobaric iodoTMT switch labeling-based mass spectrometry assay and results of MS/MS spectrum of palmitoylated peptide of GNA13. IodoTMT 6 -127 labeling on Cys14 and Cys18 (the two C in lowercase) of GNA13 peptide sequence was shown in peaks graph (upper panel). B Total, membrane (Mem) and cytosolic (Cyto) fractions of HeLa cells expressing HA-tagged WT GNA13, C14S, C18S, or C14/18S mutant of GNA13 were immunoblotted with an anti-HA antibody. α-Tubulin was used as a loading control for total cellular proteins, while Na-K-ATPase and GAPDH were used as markers of the membrane and cytosol, respectively. C HeLa cells overexpressing WT GNA13 or C14/18S mutant were incubated with cycloheximide (CHX) and analyzed by western blot at the indicated time points. D Protein levels of WT GNA13 and C14/18S mutant in HeLa cells treated with or without indicated caspase inhibitors for 24 h were detected by immunoblotting with an anti-HA antibody. α-Tubulin was used as a loading control.

Article Snippet: The cycloheximide (CHX), autophagy inhibitors, Hydroxychloroquine sulfate (HCQ), ULK-101, and the pan-caspase inhibitor Z-VAD(OMe)-FMK were purchased from TargetMol (Wellesley Hills, MA, USA).

Techniques: Labeling, Mass Spectrometry, Tandem Mass Spectroscopy, Sequencing, Membrane, Expressing, Mutagenesis, Control, Incubation, Western Blot

Journal: ACS Chemical Neuroscience

Article Title: Ester Prodrug NLRP3 Inflammasome Inhibitor NT-0796 is Brain Active due to Activation by Local Expression of Carboxylesterase‑1

doi: 10.1021/acschemneuro.5c00843

Figure Lengend Snippet: Evidence of NT-0796 hydrolysis in NHP brain homogenate. (A) NT-0796 (3 μM) was incubated with brain supernatant fraction (#1), CES1 supersomes, or buffer alone. At the indicated times, aliquots of the reaction mixtures were quenched, and concentrations of NDT-19795 were determined by LC-MS/MS analysis and indicated as a function of treatment and time at 37 °C. (B) NHP brain homogenate supernatant fractions #1 and #2, along with CES1 supersomes, were treated with FP-biotin, after which tagged proteins were subsequently captured with streptavidin agarose. After dissociation, the isolates were separated by SDS gel electrophoresis and transferred to nitrocellulose. The blot was stained sequentially with Alexafluor 680-conjugated streptavidin (red) and rabbit anti-CES1, followed by Alexafluor 800-conjugated goat-antirabbit secondary antibody (green). Lanes of the blot correspond to (1) FP-biotin-treated NHP brain supernatant fraction #1 (200 μg), (2) FP-biotin treated NHP brain supernatant fraction #2 (9 μg), (3) FP-biotin-treated CES1 supersomes (3 μg), (4) NHP brain supernatant fraction #1 (200 μg), (5) NHP brain supernatant fraction #2 (9 μg), (6) CES1 supersomes (3 μg), (7) blank, and (8) molecular weight markers (size indicated on the right). The blot was simultaneously imaged with both 700 and 800 nm lasers, allowing simultaneous visualization of both fluorescent tags.

Article Snippet: Second, the membranes were probed with streptavidin conjugated to Alexafluor 680 (Thermo Fisher, S21378; diluted 1:4000 in blocking buffer containing 1% Tween 20) for 1 h at room temperature, followed by multiple washes with PBS, 0.05% Tween 20.

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy, SDS-Gel, Electrophoresis, Staining, Molecular Weight