Discovery of Novel Adenylyl Cyclase Inhibitor by Cell-Based Screening

Abstract

We screened 2400 compounds to find novel inhibitors of the adenylyl cyclase (AC)-protein kinase A (PKA)-cAMP response-element-binding protein (CREB) signaling pathway (AC/PKA/CREB pathway). Using a multistep cell-based screening system employing split luciferase technique, we narrowed down the candidates effectively from 2400 chemical compounds and identified a novel AC inhibitor (compound 1). Since dysregulation of the AC/PKA/CREB pathway is known to cause diseases not only in the nervous system but also in other organs, compound 1 is expected to be developed as a medicine for these diseases.

What turns CREB on? And off? And why does it matter?

“…Furthermore, the binding of CREB to the CRE-DNA element can be blocked with substances binding to the DNA major groove (positively loaded substances) or directly to the bZIP of the TF (negatively loaded substances). A live imaging system using a bioluminescence-based detector system for the analysis of the interaction of KID and KIX was developed by Ishimoto and coworkers [ 92 ], which enables screening for CREB inhibitors, e.g., in herbal extracts [ 93 ].

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Identification of a Novel Protein Kinase A Inhibitor by Bioluminescence-Based Screening

“…8) In the current study, we started a new series of screening with another 2400 compounds using the same screening method. HEK293 cells stably expressing KID-KIX probe proteins were stimulated with forskolin (10 µM) in the presence of one of the compounds (10 µM) of the chemical library.…”

“…In this study, we attempt to further explore inhibitors of the AC/PKA/CREB pathway using another set of a chemical library consisting of 2400 compounds in addition to the library previously used. 8) We also tried to shorten the screening method by omitting a screening step to exclude inhibitors for wild type luciferase which was used in previous paper, since few compound inhibited wild type luciferase. 8) …”

“…8) We also tried to shorten the screening method by omitting a screening step to exclude inhibitors for wild type luciferase which was used in previous paper, since few compound inhibited wild type luciferase. 8) …”