Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The anti-cancer activity of a first-in-class small molecule targeting PCNA

doi: 10.1158/1078-0432.CCR-18-0592

Figure Lengend Snippet: (a) AOH1160 structure (patent pending). The sole substitution of an ether oxygen in AOH1160 for the corresponding methylene group in AOH39 is indicated by a red dashed box. The indicated (b) human neuroblastoma cell lines, (c) breast cancer cell lines, and (d) small cell lung cancer cell lines were cultured in the presence of various concentrations of AOH1160. The non-malignant (b) 7SM0032 cells and human PBMCs, (c) human mammary epithelial cells (hMEC), and (d) human small airway epithelial cells (SAEC) were also cultured under the same AOH1160 treatment. Cells cultured in the absence of AOH1160 were used as control. Cell growth was measured by a CellTitor Glo assay (Promega). The average of luminescence signals in triplicates normalized to the control for each cell line was graphed ± S.D. e) Three normal neural stem cell lines (NSC005-007) and three glioblastoma stem cell (GSC) lines, PBT003, PBT707, and PBT017, respectively representing three of the four glioblastoma subtypes (classical, proneural, and mesenchymal) (50) were treated with DMSO or 1 µM of AOH1160 for 72 hours. Shown are cell growth related to the control cells ± S.D. f) TRβ reporter cells were treated with various concentrations of T3, AOH39, or AOH1160 for 24 h. The effect of compounds on TRβ activity was examined by measuring the relative luminescence units (RLU) in a luminescence plate reader. Grey: Signals from T3-treated cells and black: overlapping signals from AOH39 or AOH1160-treated cells. g) Computer modeling of small molecule binding to PCNA. The model was initially built using the AAD methodology (see text) and further refined by 50ns metadynamics simulation. Shown are small molecules (in stick) and PCNA surface around the binding pocket. The loop residues of L126-Y133 of PCNA are colored in blue to green. Top panel: AOH39 is shown as colored sticks and T3 as grey sticks. Bottom panel: AOH1160 is shown as colored sticks and AOH39 as grey sticks. h) STD NMR experiments using 1 µM of PCNA. The T3 compound structure is shown on top along with proton labels. The inserted table shows the reduction in STD ± errors for each of the T3 proton positions in the presence of AOH1160 (see Methods).

Article Snippet: The human neuroblastoma cell lines: SK-N-DZ, SK-N-BE(2)c, SK-N-AS, and LAN-5, breast cancer cell lines: MDA-MB-436, MDA-MB-468, Hs578t, MCF7, HCC1937, and small cell lung cancer cell lines: H82, H524, and H526 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and were cultured in DMEM with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Cell Culture, Control, Glo Assay, Activity Assay, Microplate Reader Luminescence Measurement, Binding Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The anti-cancer activity of a first-in-class small molecule targeting PCNA

doi: 10.1158/1078-0432.CCR-18-0592

Figure Lengend Snippet: (a) Chemical structure of AOH39 (patent pending). The indicated human (b) neuroblastoma cell lines and (c) human breast cancer cell lines were cultured in the presence of various concentrations of AOH39 for 72 hours. The non-malignant 7SM0032 human embryonic progenitor cell line, (black diamonds in panel b), the immortalized, but non-transformed MCF10A cells (black circles in panel c), and PBMCs (black circles and black squares in panels b and c respectively) isolated from a healthy human donor were also cultured in the presence of the same AOH39 treatment. Cells cultured in the absence of AOH39 were used as control. Cell growth was measured by a CellTitor Glo assay (Promega). The average of luminescence signals from triplicate samples normalized to the control for each cell line was graphed plus/minus standard deviations. (d) The SK-N-DZ cells were treated with 3 µM AOH39 for 0, 6, or 24 h. Cells were fixed and stained with PI. The cellular PI fluorescence intensity was analyzed by flow cytometry. The flow cytometry data were analyzed by the FlowJo program to model various cell populations. (e) The SK-N-DZ cells were treated with 3 µM AOH39 for 0, 6, or 24 h. Total cell extracts were analyzed by western blot, using antibodies specific to γH2A.X or total H2A.X. (f) The DR-GFP and EJ5-GFP cell lines were transiently transfected with the pCBASce plasmid that expresses the I-SceI meganuclease. Three hours after transfection, cells were treated with or without 4 µM AOH39 in fresh growth medium. The HR and EJ-mediated DSB repair events, indicated by the restoration of a functional GFP gene in the respective cell lines, were quantified by measuring the relative abundance of GFP-positive cells by flow cytometry. Results from triplicates for each cell line with (light bars) or without (dark bars) AOH39 treatment were averaged and graphed plus/minus standard deviations.

Article Snippet: The human neuroblastoma cell lines: SK-N-DZ, SK-N-BE(2)c, SK-N-AS, and LAN-5, breast cancer cell lines: MDA-MB-436, MDA-MB-468, Hs578t, MCF7, HCC1937, and small cell lung cancer cell lines: H82, H524, and H526 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and were cultured in DMEM with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Cell Culture, Transformation Assay, Isolation, Control, Glo Assay, Staining, Fluorescence, Flow Cytometry, Western Blot, Transfection, Plasmid Preparation, Functional Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The anti-cancer activity of a first-in-class small molecule targeting PCNA

doi: 10.1158/1078-0432.CCR-18-0592

Figure Lengend Snippet: Synchronized (a) SK-N-BE(2)c neuroblastoma cells and (b) small cell lung cancer cells (H82 and H526) were sequentially incubated in the presence of CldU (green) and IdU (red) before and after AOH1160 treatment, respectively. Cells sequentially incubated with the same two nucleotide analogues but without AOH1160 were used as control. Shown in the left of panel (a) are representative images of the labeled DNA strands from cells treated with or without AOH1160. The lengths of CldU (green) and IdU (red) incorporated DNA segments measured from more than 30 independent DNA strands in the indicated cells treated with or without AOH1160 were averaged and graphed with standard deviations in the right of panel (a) and in panel (b). The p values were determined by the Student’s t-test on the lengths of IdU labeled DNA segments (red bars) between each AOH1160 treatment and the untreated control.

Article Snippet: The human neuroblastoma cell lines: SK-N-DZ, SK-N-BE(2)c, SK-N-AS, and LAN-5, breast cancer cell lines: MDA-MB-436, MDA-MB-468, Hs578t, MCF7, HCC1937, and small cell lung cancer cell lines: H82, H524, and H526 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and were cultured in DMEM with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Incubation, Analogues, Control, Labeling

Journal: Cancers

Article Title: EZH2 Inhibition as New Epigenetic Treatment Option for Pancreatic Neuroendocrine Neoplasms (PanNENs)

doi: 10.3390/cancers13195014

Figure Lengend Snippet: ( A ) In vitro viability curves using the metabolic surrogate assay RealTime-Glo (RTG) in 3D human primary PanNET culture treated with DMSO (control (Ctrl)) and GSK126 for 7 days. Data were first normalized per-well using a RTG baseline measurement for each individual well and then normalized to the average of the corresponding DMSO control of the respective day. Data represent means ± SEM ( n = 1 per patient, three technical replicates). RLU, relative luminescence unit. ( B ) Micro-cell-block of two representative samples. IHC of synaptophysin and H&E staining of samples from the day of isolation and DMSO-treated samples 12 days post-isolation.

Article Snippet: The RTG assay was performed according to the manufacturer’s instructions, and luminescence was measured in an Infinite ® 200 PRO plate reader (Tecan, Switzerland).

Techniques: In Vitro, Surrogate Assay, Control, Blocking Assay, Staining, Isolation

Journal: Advanced Science

Article Title: DDRGK1 Enhances Osteosarcoma Chemoresistance via Inhibiting KEAP1‐Mediated NRF2 Ubiquitination

doi: 10.1002/advs.202204438

Figure Lengend Snippet: DDRGK1 regulates NRF2 in a UFMylation‐independent pathway. A) Interaction between NRF2 and UFM1. The NRF2‐HA and ufm1‐HA plasmids were transfected into HEK293T cells. The interaction between NRF2 and ufm1 was detected by co‐immunoprecipitation with HA antibody followed by western blot with NRF2 and ufm1 antibody. B) Interaction between NRF2 and DDRGK1. The NRF2‐HA and DDRGK1‐Flag plasmids were transfected into HEK293T cells. The interaction between NRF2 and DDRGK1 was detected by co‐immunoprecipitation using anti‐HA immunomagnetic beads and anti‐Flag immunomagnetic beads. C) NRF2 levels of heart, lung, kidney, and cartilage in WT and K268R mouse detected by western blot. D) Re‐express of WT‐DDRGK1 and K267R‐DDRGK1 in DDRGK1 deficient cells rescued level of NRF2. E) Mitochondrial metabolism analysis. Cells were seeded in an XF microplate at an optimal density and cultured for 24 h. The cellular oxygen consumption rate (OCR) was measured using a Seahorse XF Analyzer. F) ROS level measurement. The controlled, DDRGK1‐knockout cells, and WT‐DDRGK1/K267R‐DDRGK1 re‐expressed cells were subjected to H 2 O 2 , then loaded with a DHE probe for 30 min and detected by flow cemetery. (MFI, mean fluorescence intensity; Three samples for each statistical analysis **** p < 0.0001).

Article Snippet: Then, 10 μL of CCK8 reagent was added, maintained at 37 °C for 1 h. The optical density was then measured at 450 nm using an automatic i‐control microplate reader (Tecan Life Sciences, Switzerland).

Techniques: Transfection, Immunoprecipitation, Western Blot, Cell Culture, Knock-Out, Fluorescence

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: Cascade assay for target engagement confirms the mechanisms of action for reference compounds. (A) Schematic of MK2/3 signaling. (B) Isolated reactions tested in the biochemical cascade assays. (C) Concentration-response profiles for CC-99677 and ATI-450 against active kinases on their own (open symbols) or for cascade assays using active p38α to activate unactive MK2 or MK3 (solid symbols). Phosphorylation of the respective substrates for the individual enzymes, or in the cascade assays for the unactive enzyme (MK2 or MK3), were measured by HTRF.

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: Drug discovery, Isolation, Concentration Assay, Phospho-proteomics

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: Dual HTRF assay development. (A) Schematic of the dual HTRF assay targeting free MK2 bound to a covalent biotinylated tracer (biotin|cov; see panel (C)). (B) Determination of specific antibody pairs for detecting total MK2. Competition (left) of unlabeled monoclonal antibodies for the epitope of anti-MK2 D1E11 terbium (Tb)-cryptate, enabling the identification of a minimally competitive antibody for quantification of total MK2. Unlabeled antibodies were titrated into reactions containing 0.5× anti-MK2 D1E11 Tb-cryptate and 10 nM biotin|cov tracer paired with streptavidin Alexa Fluor 488. Mouse anti-MK2 7H4.2 was chosen for further assay development and directly conjugated to Alexa Fluor 633 for enhanced HTRF signal. Specificity of this antibody pair for MK2 was tested against recombinant human MK2 or MK3 (right). The antibody pair anti-MK3 D54E4 Tb-cryptate with anti-MK3 2B5 Alexa Fluor 568 was used as a positive control for detection of MK3. Note the cross-reactivity of anti-D1E11 Tb-cryptate for MK3. (C) Structure of biotin|cov, the tracer used for the dual HTRF assays (left). The specificity of this tracer for recombinant human MK2 over MK3 is shown in the associated plot (right), using 0.5× anti-MK2 D1E11-Tb cryptate as the energy donor. (D) CC-99677 target engagement on 10 nM recombinant human MK2 using the dual HTRF assay reagents (anti-MK2 pair plus biotin|cov with streptavidin Alexa Fluor 488). The cause of the observed incomplete target occupancy by this compound is unknown. (E) Quantification of endogenous MK2 protein abundance in human cell lines (left) and murine RAW264.7 cells or splenocytes (right). Specificity of the chosen anti-MK2 pair was demonstrated by the absence of signal from genetic knockout samples (human U937 and mouse splenocytes). HCC1428 possessed the highest detectable concentration of MK2 among the adherent human cell lines (family of red symbols), so it was used for studies to measure endogenous target engagement.

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: HTRF Assay, Bioprocessing, Assay Development, Recombinant, Positive Control, Drug discovery, Quantitative Proteomics, Knock-Out, Concentration Assay

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: NanoBRET assay development. (A) Emission spectrum of NanoLuciferase measured live in dimethyl sulfoxide-treated HeLa cells over-expressing either amino- or carboxy-terminal MK2 fusions, with or without 0.17 mM (0.004%) Triton X-100 permeabilization (dashed or solid lines, respectively). The filter band passes used to measure NanoBRET signal are highlighted (centered at 452 nm for NanoLuciferase and 600 nm for BODIPY585). Magnified portion of the spectrum (right) shows the fraction of light from the NanoLuciferase donor that overlaps with –and should be subtracted from– the BODIPY acceptor emission signal. (B) Characterization of BODIPY|rev and BODIPY585|cov tracers, which compete for the same binding pocket as CC-99677. Structures of tracers (left) are shown alongside magnified emission spectra when added to HeLa cells transfected with plasmids encoding either amino- or carboxy-terminal MK2 fusions (middle) as well as the ratiometric NanoBRET signal following a titration of each tracer (right). Signal was measured 1 hour after tracer addition to the live intact (solid) or permeabilized (open/dashed line) cells using a CLARIOstar Plus (average of 5 replicates with standard deviation is shown). Line weight and style follow the legend in panel (A). (C) and (D) NanoBRET signal kinetics in the presence of BODIPY585|cov tracer, using well-by-well acquisitions (C) on the CLARIOstar Plus (CLARIOstar) or full-plate imaging (D) with a GNF Systems luminescence plate reader (LPR). NanoLuciferase signal (blue profiles in shaded background) decayed over time whereas the NanoBRET ratio (red profiles in white background) increased with exposure time to tracer. Continuous measurements for longer than 1 hour were achieved by supplementing with Endurazine substrate. (E) and (F) Measurement of k -on (E) and k -off (F) for CC-99677 using NanoBRET with BODIPY585|cov on a luminescence plate reader. The time points denote when CC-99677-containing medium was exchanged for medium containing excess BODIPY585|cov, which irreversibly quenched the system by saturating free binding sites. The profile for a reversible compound that occupies the same pocket as the covalent CC-99677 (gray, open circles) is included in the k -off plots (F).

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: Assay Development, Expressing, Binding Assay, Transfection, Titration, Standard Deviation, Imaging, Microplate Reader Luminescence Measurement

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: Comparison of compound target occupancy assays. (A) to (C) Measurement of MK2 target occupancy using dual HTRF, for endogenous protein targeting, in human HCC1428 cells (A) or murine RAW264.7 cells (B) versus NanoBRET in HeLa cells transfected with NanoLuciferase amino-terminally fused to human MK2 (C). Cells were treated with compound for 3 hours before washout. Curve fits are shown for the “free” signal (the ratio of “tracer”/“total”) for CC-99677 treatment, with regression ( R 2 ) values of 0.96 (RAW264.7 in HTRF); 0.92 (HCC1428 in HTRF), and 0.99 (HeLa in NanoBRET). (D) and (E) NanoBRET target occupancy data for 3-hour CC-99677 exposure in the presence of increasing concentrations of fetal bovine or human serum (D) to model in vivo compound availability using the NanoBRET assay. The right-shifted phenotype of CC-99677 is independent of human serum lot (E), suggesting that the free-fraction of CC-99677 compound will be reduced in circulation.

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: Comparison, Transfection, In Vivo