A Cell-based beta-Lactamase Reporter Gene Assay for the CREB Signaling Pathway.

Area of Science:

• Molecular Biology
• Neuroscience
• Pharmacology

Background:

• Cyclic-AMP Response Element Binding (CREB) proteins are transcription factors regulating gene expression via cAMP Response Elements (CREs).
• CREB signaling is crucial for physiological processes like long-term memory.
• Enhancing CREB signaling is a potential therapeutic strategy for memory disorders.

Purpose of the Study:

• To identify small molecule compounds that enhance CREB pathway signaling.
• To optimize and validate a cell-based beta-lactamase reporter gene assay for CREB pathway screening.
• To utilize a quantitative high throughput screening (qHTS) platform for compound discovery.

Main Methods:

• A cell-based beta-lactamase reporter gene assay for the CREB pathway was optimized and validated in a 1536-well plate format.
• The LOPAC library (1280 compounds) was screened in triplicate using a quantitative high throughput screening (qHTS) platform.
• Active compounds were identified by their effect on CREB pathway signaling.

Main Results:

• The assay successfully identified known modulators of the CREB pathway, validating its performance.
• Twelve known phosphodiesterase (PDE) inhibitors were identified as active compounds.
• Forskolin, an adenylate cyclase activator, was also identified, further confirming assay validity.

Conclusions:

• The validated qHTS assay is effective for identifying novel small molecule CREB signaling enhancers.
• These enhancers can aid in the chemical genetic dissection of the CREB pathway.
• The identified compounds serve as potential starting points for developing memory-enhancing therapeutics.