Reporter system for high throughput screening of compounds and uses thereof

摘要

The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. To directly monitor stabilization of Nrf2 we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). It is shown herein that Neh2 domain is sufficient for recognition, ubiquitination and proteasomal degradation of Neh2-luciferase fusion protein. The novel Neh2-luc reporter system allows direct monitoring of the adaptive response to redox stress and classification of drugs based on the time-course of reporter activation. The novel reporter was used to screen a library of compounds to identify activators of Nrf2. The most robust and yet non toxic Nrf2 activators found—nordihydroguaiaretic acid, fisetin, and gedunin-induced astrocyte-dependent neuroprotection from oxidative stress via an Nrf2-dependent mechanism.

主权项

1. A nucleic acid construct, comprising a nucleotide sequence which codes for a fusion protein which comprises an Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-ECH homologous (Neh2) domain linked to a reporter, wherein the Neh2 domain consists of an amino acid sequence having at least 95% identity with SEQ ID NO: 11.