Novel Approach to High Throughput Screening for Activators of Transcription Factors

Smirnova, Natalia; Hushpulian, Dmitry; Ratan, Rajiv R.; Gazaryan, Irina G.

doi:10.5772/28743

Cited by 1 publication

(1 citation statement)

References 47 publications

(55 reference statements)

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“…In most cases, specific information related to the reporter system and responsive cell lines is limited, which means that researchers who wish to identify chemical compounds that modulate the activity of specific transcription factors must also carry out laborious optimization studies. We surmise that this may be the reason that elaborate reporter systems for the screening of HIF modulating chemicals are currently limited (Smirnova et al 2011). Since phenotype screening, including transactivating reporter gene assays, is an important approach to identify lead compounds, in this study we aimed to develop an effective and highly sensitive screening system applicable for the high-throughput screening (HTS) of lead compounds for the hypoxia-response.…”

Section: Introductionmentioning

confidence: 99%

Hypoxia-Sensitive Reporter System for High-Throughput Screening

Tsujita

Kawaguchi

Dan

et al. 2015

Tohoku J. Exp. Med.

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The induction of anti-hypoxic stress enzymes and proteins has the potential to be a potent therapeutic strategy to prevent the progression of ischemic heart, kidney or brain diseases. To realize this idea, small chemical compounds, which mimic hypoxic conditions by activating the PHD-HIF-α system, have been developed. However, to date, none of these compounds were identified by monitoring the transcriptional activation of hypoxia-inducible factors (HIFs). Thus, to facilitate the discovery of potent inducers of HIF-α, we have developed an effective high-throughput screening (HTS) system to directly monitor the output of HIF-α transcription. We generated a HIF-α-dependent reporter system that responds to hypoxic stimuli in a concentration-and time-dependent manner. This system was developed through multiple optimization steps, resulting in the generation of a construct that consists of the secretion-type luciferase gene (Metridia luciferase, MLuc) under the transcriptional regulation of an enhancer containing 7 copies of 40-bp hypoxia responsive element (HRE) upstream of a mini-TATA promoter. This construct was stably integrated into the human neuroblastoma cell line, SK-N-BE(2)c, to generate a reporter system, named SKN:HRE-MLuc. To improve this system and to increase its suitability for the HTS platform, we incorporated the next generation luciferase, Nano luciferase (NLuc), whose longer half-life provides us with flexibility for the use of this reporter. We thus generated a stably transformed clone with NLuc, named SKN:HRE-NLuc, and found that it showed significantly improved reporter activity compared to SKN:HRE-MLuc. In this study, we have successfully developed the SKN:HRE-NLuc screening system as an efficient platform for future HTS.

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Section: Introductionmentioning

confidence: 99%