Limits...
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.


Dose–response curves. (a) The non-specific response of Dianiline across five concentrations. (b) The miRNA-182 specific response of Panobinostat. (c) The miRNA-183 specific response of AHPN.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2-0271678X15609939: Dose–response curves. (a) The non-specific response of Dianiline across five concentrations. (b) The miRNA-182 specific response of Panobinostat. (c) The miRNA-183 specific response of AHPN.

Mentions: The ultimate goal of this work was to develop a system capable of identifying molecular entities (MEs)/active pharmaceutical ingredients (APIs) capable of upregulating global SUMOylation through the inhibition of miRNAs 182 and/or 183. To minimize interference and increase our confidence in hits ascertained during screening, we designed constructs that contain two different reporters (dual reporter system), firefly luciferase and Renilla luciferase (Figure 1), which are not homologous and therefore have unrelated bioluminescent properties. We confirmed that the presence of mature miR-182 or miR-183 would lead to a decrease in luciferase (both firefly and Renilla) signal, enabling the detection of putative miR-182 (or miR-183) levels (Supplementary Figure 2). We then established cell lines which stably expressed these constructs as described in Figure 1, and used them for the screening of small molecule libraries in a 1536 well format. From a total of 4489 compounds screened, 120 compounds were initially identified in the course of the primary screening process. These 120 compounds were subsequently selected and re-screened for validation through the use of confirmatory assays. From the follow-up screening, 21 active compounds (listed in Table 1) were confirmed based on their activities in both firefly and Renilla luminescence assays and were taken forward for further study/characterization. We note that most confirmed compounds do not give equivalent percentage changes in signal in both channels, perhaps due to a difference in sensitivity in the detection methodology for these readouts (Figure 2 and Supplementary Figure 3).Table 1.


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
Dose–response curves. (a) The non-specific response of Dianiline across five concentrations. (b) The miRNA-182 specific response of Panobinostat. (c) The miRNA-183 specific response of AHPN.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2-0271678X15609939: Dose–response curves. (a) The non-specific response of Dianiline across five concentrations. (b) The miRNA-182 specific response of Panobinostat. (c) The miRNA-183 specific response of AHPN.
Mentions: The ultimate goal of this work was to develop a system capable of identifying molecular entities (MEs)/active pharmaceutical ingredients (APIs) capable of upregulating global SUMOylation through the inhibition of miRNAs 182 and/or 183. To minimize interference and increase our confidence in hits ascertained during screening, we designed constructs that contain two different reporters (dual reporter system), firefly luciferase and Renilla luciferase (Figure 1), which are not homologous and therefore have unrelated bioluminescent properties. We confirmed that the presence of mature miR-182 or miR-183 would lead to a decrease in luciferase (both firefly and Renilla) signal, enabling the detection of putative miR-182 (or miR-183) levels (Supplementary Figure 2). We then established cell lines which stably expressed these constructs as described in Figure 1, and used them for the screening of small molecule libraries in a 1536 well format. From a total of 4489 compounds screened, 120 compounds were initially identified in the course of the primary screening process. These 120 compounds were subsequently selected and re-screened for validation through the use of confirmatory assays. From the follow-up screening, 21 active compounds (listed in Table 1) were confirmed based on their activities in both firefly and Renilla luminescence assays and were taken forward for further study/characterization. We note that most confirmed compounds do not give equivalent percentage changes in signal in both channels, perhaps due to a difference in sensitivity in the detection methodology for these readouts (Figure 2 and Supplementary Figure 3).Table 1.

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.