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1.
Figure 5

Figure 5. From: Identification through high-throughput screening of 4'-methoxyflavone and 3',4'-dimethoxyflavone as novel neuroprotective inhibitors of parthanatos.

Customized flavonoid library for structure–activity relationship study. The library contains 27 compounds, consisting of 2 non-flavone polyphenolic compounds, the parent compound (flavone), 8 compounds carrying only hydroxy-substitutions on the parent compound, 3 compounds with both hydroxy- and methoxy-substitutions, and 13 compounds carrying only methoxy-substitutions on the parent flavone. The substitutions are in varied positions on the ring and are to different degrees. The lead compound, 4MF, is included as an internal control (serial number 18, highlighted). These compounds were initially prepared in DMSO as 10 mM stock solutions, from which 2 mM working stock solutions for screening were prepared.

A A Fatokun, et al. Br J Pharmacol. 2013 July;169(6):1263-1278.
2.
Figure 6

Figure 6. From: Identification through high-throughput screening of 4'-methoxyflavone and 3',4'-dimethoxyflavone as novel neuroprotective inhibitors of parthanatos.

Customized library screen. (A) Model screening assay using luminescence-based CTG for the quantification of cell viability. MNNG reduced cell viability significantly. DPQ protected against the toxic MNNG effect, but z-VAD did not. Quantification was done 15–20 h after 25 min exposure to MNNG followed by restoration to growth medium. (B) Customized library compounds screened at 10 μM against MNNG in HeLa cells. (C) Customized library compounds screened at 10 μM against hydrogen peroxide (H2O2) in HeLa cells. (D) Customized library compounds screened at 10 μM against MNNG in dopaminergic SH-SY5Y cells. Arrows, arrowheads or inscriptions show, as indicated, the lack of effect of the parent compound, flavone, the effect of 4MF, or the effect of a second protective compound, DMF. ***P < 0.001 compared with control; ###P < 0.001 compared with MNNG alone. Data in A represent an average of four independent experiments, while B–D show representative data for experiments conducted twice, with similar results obtained in both cases.

A A Fatokun, et al. Br J Pharmacol. 2013 July;169(6):1263-1278.
3.
Figure 3

Figure 3. From: Identification through high-throughput screening of 4'-methoxyflavone and 3',4'-dimethoxyflavone as novel neuroprotective inhibitors of parthanatos.

High-throughput screen of chemical libraries. Screening data for (A) MSSPL and (B) JHDL. MSSPL contains 2000 compounds in 25 plates while JHDL has 3120 compounds in 42 plates, making a total of 5120 compounds that were screened. MSSPL was screened robotically while JHDL was screened manually. Values shown represent normalized values after the effect of the toxic stimulus MNNG was set to 40% of the normal control for each plate. A hit was taken as a compound giving a normalized value of at least 60% (indicated by the solid horizontal line in Figure A or Figure B). (C) Pooled data for plates in the MSSPL confirming overall screening replication of the model assay for 22 plates (three plates, numbers 19, 21 and 25, that required rescreening, were excluded). (D) Pooled data for all 42 plates in the JHDL confirming overall screening replication of the model assay. The average Z' factor for each library was 0.7. ***P < 0.001 compared with control; ###P < 0.001 compared with MNNG alone.

A A Fatokun, et al. Br J Pharmacol. 2013 July;169(6):1263-1278.
4.
Figure 1

Figure 1. From: Identification through high-throughput screening of 4'-methoxyflavone and 3',4'-dimethoxyflavone as novel neuroprotective inhibitors of parthanatos.

Flow chart for the screening project. Assay was developed and optimized in HeLa cells and then employed for high-throughput screen of two chemical libraries: the MSSP Library and the JHDL. AB (fluorescence-based) or CTG (luminescence-based) was used to quantify cell viability. Compounds showing toxicity were rescreened at 2 and 5 μM while those with marginal protective effects were rescreened at 20 μM. The 10 hits from the primary screen were rescreened for validation. One compound was confirmed as true hit. A number of small molecules belonging to the same class as this compound or similar classes were then selected and screened, resulting in one more hit that was then validated. Dose–response data were obtained for the two hits; they were proven to block the synthesis and accumulation of toxic PAR polymer and were also neuroprotective in cell culture. They are now to be investigated for their ability to afford neuroprotection in animal models of neuronal death.

A A Fatokun, et al. Br J Pharmacol. 2013 July;169(6):1263-1278.
5.
Figure 2

Figure 2. From: Identification through high-throughput screening of 4'-methoxyflavone and 3',4'-dimethoxyflavone as novel neuroprotective inhibitors of parthanatos.

Assay development and optimization for high-throughput screen. (A) Model assay for the screening project. MNNG reduces cell viability significantly, protected against by DPQ (positive control), but not by Z-VAD-fmk (negative control), although neither control had any effect on its own. Viability was quantified 15–20 h after restoration of cultures to growth medium following 25 min exposure to MNNG (50 μM). (B) MNNG reduces cell viability in a concentration- and time-dependent manner, up to 20 h of recovery. (C) Hydrogen peroxide reduces viability in a concentration and time-dependent manner, up to 2 h of exposure. (D) Replication of model assay with positive and negative controls present before, during and after MNNG treatment (viability was quantified 15–20 h post recovery). (E) Replication of model assay with positive and negative controls present during and after MNNG treatment. (F) Replication of model assay with positive and negative controls present only after MNNG treatment. Values shown inside columns (D–F) are normalized, with control viability taken as 100%. Optimization was conducted in HeLa cells and viability was quantified by AB. **P < 0.01, ***P < 0.001 compared with the control; ###P < 0.001; ns, not significant compared with the effect of MNNG alone. Data shown represent the average of at least four separate experiments or replicates.

A A Fatokun, et al. Br J Pharmacol. 2013 July;169(6):1263-1278.
6.
Figure 7

Figure 7. From: Identification through high-throughput screening of 4'-methoxyflavone and 3',4'-dimethoxyflavone as novel neuroprotective inhibitors of parthanatos.

Comparative concentration–response data and inhibition of PAR synthesis and accumulation by 4MF and DMF. (A) Graph showing the concentration-dependent protective effects of 4MF and DMF against MNNG in HeLa cells, with viability quantified by CTG. Values shown are averages of two separate experiments (100 μM shown to demonstrate that effects of the compounds peaked at 25 μM). (B) Concentration–response data in (A) transformed into a sigmoid curve for the determination of EC50. The EC50 values are 11.41 ± 1.04 μM and 9.94 ± 1.05 μM, for 4MF and DMF respectively (GraphPad Prism 5.03). (C, D) Assessment of the effects of 4MF and DMF on the levels of PAR polymer induced by MNNG in HeLa cells. Cultures were treated with MNNG (controls treated with DMSO) for 5 min in the presence or absence of increasing concentrations of 4MF or DMF. They were then probed with anti-PAR rabbit polyclonal primary antibody (1:10 000; Trevigen) and, after washing, incubated with anti-rabbit IgG (whole molecule) – peroxidase secondary antibody (1:5000; Sigma). Detection was by chemiluminescence with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and immunoblots were visualized on X-ray films. β-Actin was used as a loading control. Data shown are representative of experiments that were repeated at least three times with similar results.

A A Fatokun, et al. Br J Pharmacol. 2013 July;169(6):1263-1278.
7.
Figure 4

Figure 4. From: Identification through high-throughput screening of 4'-methoxyflavone and 3',4'-dimethoxyflavone as novel neuroprotective inhibitors of parthanatos.

Hit identification and confirmation. (A) Compounds from the two primary screens showing MNNG value-normalized percentage viability of at least 60%. Ten (10) such compounds were identified, seven (7) from the MSSPL (thick, black horizontal line) and three (3) from the JHDL (thick, grey horizontal line). The 40% dotted horizontal line represents normalized MNNG value while the 60% dotted horizontal line represents the minimal level of protection required for a compound to be classified as a hit. Compounds on or above the 60% line were taken as hits. Only one out of the ten compounds, 4MF, was confirmed as a true hit. (B) Chemical structure of 4MF, showing methoxylation (–OCH3) of the parent flavone structure at the 4′ position of the heterocycle. (C) 4MF protects against toxic MNNG effect in a concentration-dependent manner, reaching significance and peaking at 25 μM. Concentrations were chosen in twofold increments up to 100 μM. Numerals on the horizontal axis represent 4MF (2F08) concentrations in μM (100 μM included to demonstrate that effect peaked at 25 μM). (D) Concentration-response data transformed to a sigmoid curve for the determination of EC50 value of 4MF (2F08), calculated as 10.41 ± 1.31 μM (GraphPad Prism 5.03; GraphPad Software Inc., San Diego, CA, USA). (E) Chemical structure of the parent compound, flavone. (F) Chemical structure of DMF, showing methoxylation of the parent flavone structure at the 3' and 4′ positions. Viability was quantified by AB. Values shown in (C) or (D) are for four separate experiments. ***P < 0.001 compared with control; #P < 0.05, ##P < 0.01 compared with MNNG alone.

A A Fatokun, et al. Br J Pharmacol. 2013 July;169(6):1263-1278.
8.
Figure 8

Figure 8. From: Identification through high-throughput screening of 4'-methoxyflavone and 3',4'-dimethoxyflavone as novel neuroprotective inhibitors of parthanatos.

4MF and DMF protect against neuronal death induced by NMDA. Primary neuronal cultures were prepared from gestational day 15–16 fetal CD1 mice. Inhibition of glial proliferation was achieved by addition to the growth medium of 5-fluoro-2'-deoxyuridine (5F2DU, Sigma, 30 μM) at 3–4 days in vitro. Cultures so treated represent at least 80% neurones. Cultures were used for experiments at 12–14 days in vitro. Control cultures were treated with CSS. NMDA was always freshly prepared in CSS, with 10 μM glycine added to act as a co-agonist at the NMDA receptor. Each treatment was for 5 min. Cultures were then restored to their conditioned growth medium for the next 15–20 h. To assess cell death, cultures were stained with Hoechst 33342 (5 mg·mL−1, labels all cells blue) for 5 min and propidium iodide (1 mg·mL−1, labels dead cells red) for a further 5 min. Medium was then aspirated and cultures washed with CSS before image acquisition after placing the plates on a motorized stage of a Zeiss microscope. Computer-assisted cell counting was then performed with the aid of specialized software (Axiovision 4.6, Zeiss, Germany). At least three separate fields were counted for each treatment. Cell death was calculated as the ratio of PI-stained (red) cells to Hoechst 33342-stained (blue) cells, expressed as a percentage. (A) 4MF concentration-dependently reduced neuronal death induced by exposure to NMDA. (B) Photomicrographs showing dead- and live-cell staining to indicate the death-inducing effect of NMDA on neurones and the concentration-dependent neuroprotective effect of 4MF. Neuroprotection is clearly evident even from the lowest 4MF concentration tested. (C) DMF concentration-dependently reduced neuronal death induced by exposure to NMDA but was less potent than 4MF. (D) Photomicrographs showing dead- and live-cell staining to indicate the death-inducing effect of NMDA on neurones and the concentration-dependent neuroprotective effect of DMF. Neuroprotection is evident with DMF concentration of 25 μM or higher. Data shown are an average of three different fields and are representative of at least three separate experiments with similar results. ***P < 0.001 compared with CSS control; ##P < 0.01, ###P < 0.001 compared with NMDA alone.

A A Fatokun, et al. Br J Pharmacol. 2013 July;169(6):1263-1278.

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