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A novel quantitative high-throughput screen identifies drugs that both activate SUMO conjugation via the inhibition of microRNAs 182 and 183 and facilitate neuroprotection in a model of oxygen and glucose deprivation

View Article: PubMed Central - PubMed

ABSTRACT

The conjugation/de-conjugation of Small Ubiquitin-like Modifier (SUMO) has been shown to be associated with a diverse set of physiologic/pathologic conditions. The clinical significance and ostensible therapeutic utility offered via the selective control of the global SUMOylation process has become readily apparent in ischemic pathophysiology. Herein, we describe the development of a novel quantitative high-throughput screening (qHTS) system designed to identify small molecules capable of increasing SUMOylation via the regulation/inhibition of members of the microRNA (miRNA)-182 family. This assay employs a SHSY5Y human neuroblastoma cell line stably transfected with a dual firefly-Renilla luciferase reporter system for identification of specific inhibitors of either miR-182 or miR-183. In this study, we have identified small molecules capable of inducing increased global conjugation of SUMO in both SHSY5Y cells and rat E18-derived primary cortical neurons. The protective effects of a number of the identified compounds were confirmed via an in vitro ischemic model (oxygen/glucose deprivation). Of note, this assay can be easily repurposed to allow high-throughput analyses of the potential drugability of other relevant miRNA(s) in ischemic pathobiology.

No MeSH data available.


Treatments with small molecules identified by qHTS increase the levels of SUMO conjugation and the Ubc9 conjugase in SHSY5Y parent cells. (a) Representative immunoblots of high molecular weight (>100 kDa) SUMO-1 and SUMO-2,3 conjugates and the Ubc9 protein in the total cell lysates from SHSY5Y cells treated with various compounds with indicated concentrations for 13.5 h. (b) Quantitative analyses of the conjugates and Ubc9 from three independent experiments. High molecular weight SUMO-1 or SUMO-2,3 conjugates (>100 kDa) were cropped in each lane and the total intensity measured. The densities were normalized to corresponding actin levels and expressed as the ratio to control (DMSO alone). Data represent the mean+/−standard deviation of three independent experiments. **p < 0.01, *p < 0.05 compared to DMSO control.
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fig3-0271678X15609939: Treatments with small molecules identified by qHTS increase the levels of SUMO conjugation and the Ubc9 conjugase in SHSY5Y parent cells. (a) Representative immunoblots of high molecular weight (>100 kDa) SUMO-1 and SUMO-2,3 conjugates and the Ubc9 protein in the total cell lysates from SHSY5Y cells treated with various compounds with indicated concentrations for 13.5 h. (b) Quantitative analyses of the conjugates and Ubc9 from three independent experiments. High molecular weight SUMO-1 or SUMO-2,3 conjugates (>100 kDa) were cropped in each lane and the total intensity measured. The densities were normalized to corresponding actin levels and expressed as the ratio to control (DMSO alone). Data represent the mean+/−standard deviation of three independent experiments. **p < 0.01, *p < 0.05 compared to DMSO control.

Mentions: To determine the effect of the active compounds identified via the primary screen on the induction of SUMO conjugation, the 21 compounds were tested in orthogonal cell-based assays with SHSY5Y cells. The compounds were incubated with SHSY5Y cells for 13.5 h at both a low and high concentration extrapolated from the concentration–response curves of the confirmatory assay. As shown in Figure 3, immunoblotting effectively demonstrated that the majority of the compounds identified were indeed capable of upregulating SUMO1 and SUMO-2/3 conjugation, thereby confirming the biologic validity of the positive hits resulting from the qHTS assay. Further, it was noted that many of the compounds seemed to increase the levels of the sole SUMO E2 conjugase Ubc9 as well (Figure 3). To examine whether the upregulation of global SUMO conjugation by these compounds in SHSY5Y cells (stable derivatives of which we used for the screening) were cell type specific, or not, we next examined the effect of the compounds on the SUMOylation levels of primary cortical neurons isolated from rat embryos. Since the lower dose of the majority of the compounds had a similar effect when compared to the higher dose in SHSY5Y cells (Figure 3), we explored the lower/physiologically compatible dose in primary cortical neurons. As shown in Figure 4, most compounds were capable of increasing the levels of global SUMO conjugation in primary cortical neurons. Interestingly, the levels of global SUMOylation induced did vary from compound to compound between the SHSY5Y cell line and the primary cortical neurons (Figures 3 and 4). To exclude the confounding contributions of compound cytotoxicity, a cell viability assay was performed in parallel with the SUMO conjugation assay. We found that the increases in global SUMOylation were unrelated to a cellular stress response or cell death as compound cytotoxicity was not observed; critically, cells were treated with the compounds for an equivalent amount of time (Supplementary Figure 4).Figure 3.


A novel quantitative high-throughput screen identifies drugs that both activate SUMO conjugation via the inhibition of microRNAs 182 and 183 and facilitate neuroprotection in a model of oxygen and glucose deprivation
Treatments with small molecules identified by qHTS increase the levels of SUMO conjugation and the Ubc9 conjugase in SHSY5Y parent cells. (a) Representative immunoblots of high molecular weight (>100 kDa) SUMO-1 and SUMO-2,3 conjugates and the Ubc9 protein in the total cell lysates from SHSY5Y cells treated with various compounds with indicated concentrations for 13.5 h. (b) Quantitative analyses of the conjugates and Ubc9 from three independent experiments. High molecular weight SUMO-1 or SUMO-2,3 conjugates (>100 kDa) were cropped in each lane and the total intensity measured. The densities were normalized to corresponding actin levels and expressed as the ratio to control (DMSO alone). Data represent the mean+/−standard deviation of three independent experiments. **p < 0.01, *p < 0.05 compared to DMSO control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2 - License 3
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getmorefigures.php?uid=PMC4759677&req=5

fig3-0271678X15609939: Treatments with small molecules identified by qHTS increase the levels of SUMO conjugation and the Ubc9 conjugase in SHSY5Y parent cells. (a) Representative immunoblots of high molecular weight (>100 kDa) SUMO-1 and SUMO-2,3 conjugates and the Ubc9 protein in the total cell lysates from SHSY5Y cells treated with various compounds with indicated concentrations for 13.5 h. (b) Quantitative analyses of the conjugates and Ubc9 from three independent experiments. High molecular weight SUMO-1 or SUMO-2,3 conjugates (>100 kDa) were cropped in each lane and the total intensity measured. The densities were normalized to corresponding actin levels and expressed as the ratio to control (DMSO alone). Data represent the mean+/−standard deviation of three independent experiments. **p < 0.01, *p < 0.05 compared to DMSO control.
Mentions: To determine the effect of the active compounds identified via the primary screen on the induction of SUMO conjugation, the 21 compounds were tested in orthogonal cell-based assays with SHSY5Y cells. The compounds were incubated with SHSY5Y cells for 13.5 h at both a low and high concentration extrapolated from the concentration–response curves of the confirmatory assay. As shown in Figure 3, immunoblotting effectively demonstrated that the majority of the compounds identified were indeed capable of upregulating SUMO1 and SUMO-2/3 conjugation, thereby confirming the biologic validity of the positive hits resulting from the qHTS assay. Further, it was noted that many of the compounds seemed to increase the levels of the sole SUMO E2 conjugase Ubc9 as well (Figure 3). To examine whether the upregulation of global SUMO conjugation by these compounds in SHSY5Y cells (stable derivatives of which we used for the screening) were cell type specific, or not, we next examined the effect of the compounds on the SUMOylation levels of primary cortical neurons isolated from rat embryos. Since the lower dose of the majority of the compounds had a similar effect when compared to the higher dose in SHSY5Y cells (Figure 3), we explored the lower/physiologically compatible dose in primary cortical neurons. As shown in Figure 4, most compounds were capable of increasing the levels of global SUMO conjugation in primary cortical neurons. Interestingly, the levels of global SUMOylation induced did vary from compound to compound between the SHSY5Y cell line and the primary cortical neurons (Figures 3 and 4). To exclude the confounding contributions of compound cytotoxicity, a cell viability assay was performed in parallel with the SUMO conjugation assay. We found that the increases in global SUMOylation were unrelated to a cellular stress response or cell death as compound cytotoxicity was not observed; critically, cells were treated with the compounds for an equivalent amount of time (Supplementary Figure 4).Figure 3.

View Article: PubMed Central - PubMed

ABSTRACT

The conjugation/de-conjugation of Small Ubiquitin-like Modifier (SUMO) has been shown to be associated with a diverse set of physiologic/pathologic conditions. The clinical significance and ostensible therapeutic utility offered via the selective control of the global SUMOylation process has become readily apparent in ischemic pathophysiology. Herein, we describe the development of a novel quantitative high-throughput screening (qHTS) system designed to identify small molecules capable of increasing SUMOylation via the regulation/inhibition of members of the microRNA (miRNA)-182 family. This assay employs a SHSY5Y human neuroblastoma cell line stably transfected with a dual firefly-Renilla luciferase reporter system for identification of specific inhibitors of either miR-182 or miR-183. In this study, we have identified small molecules capable of inducing increased global conjugation of SUMO in both SHSY5Y cells and rat E18-derived primary cortical neurons. The protective effects of a number of the identified compounds were confirmed via an in&nbsp;vitro ischemic model (oxygen/glucose deprivation). Of note, this assay can be easily repurposed to allow high-throughput analyses of the potential drugability of other relevant miRNA(s) in ischemic pathobiology.

No MeSH data available.