ABSTRACT
The Keap1 (Kelch-like ECH-associated protein 1)-Nrf2 (nuclear factor erythroid 2-related factor 2)-ARE (antioxidant response element) pathway is the major defending mechanism against oxidative stresses, and directly disrupting the Keap1-Nrf2 protein-protein interaction (PPI) has been an attractive strategy to target oxidative stress-related diseases, including cardiovascular diseases. Here, we describe the design, synthesis, and structure-activity relationships (SARs) of indoline-based compounds as potent Keap1-Nrf2 PPI inhibitors. Comprehensive SAR analysis and thermodynamics-guided optimization identified 19a as the most potent inhibitor in this series, with an IC50 of 22 nM in a competitive fluorescence polarization assay. Further evaluation indicated the proper drug-like properties of 19a. Compound 19a dose-dependently upregulated genes and protein level of Nrf2 as well as its downstream markers and showed protective effects against lipopolysaccharide-induced injury in both H9c2 cardiac cells and mouse models. Collectively, we reported here a novel indoline-based Keap1-Nrf2 PPI inhibitor as a potential cardioprotective agent.
Subject(s)
Cardiotonic Agents/pharmacology , Drug Design , Indoles/chemistry , Kelch-Like ECH-Associated Protein 1/metabolism , NF-E2-Related Factor 2/metabolism , Humans , Kelch-Like ECH-Associated Protein 1/antagonists & inhibitors , NF-E2-Related Factor 2/antagonists & inhibitors , Protein Binding , Structure-Activity Relationship , ThermodynamicsABSTRACT
Reversibly altering endogenous protein levels are persistent issues. Herein, we designed photoswitchable azobenzene-proteolysis targeting chimeras (Azo-PROTACs) by including azobenzene moieties between ligands for the E3 ligase and the protein of interest. Azo-PROTACs are light-controlled small-molecule tools for protein knockdown in cells. The light-induced configuration change can switch the active state to induce protein degradation activity, which can be reversely controlled by light exposure in intact cells. We compared the protein degradation abilities of Azo-PROTACs with different configurations and linker lengths. Using the stable form with the best degradation ability against the BCR-ABL fusion and ABL proteins in myelogenous leukemia K562 cells, we showed that Azo-PROTAC combines the potent protein knockdown and facile cell uptake properties of the small-molecule PROTAC with a reversible photoswitchability, offering a promising chemical knockdown strategy based on the light-induced reversible on/off properties.
Subject(s)
Azo Compounds/pharmacology , Dasatinib/analogs & derivatives , Dasatinib/pharmacology , Lenalidomide/analogs & derivatives , Lenalidomide/pharmacology , Adaptor Proteins, Signal Transducing/metabolism , Azo Compounds/chemical synthesis , Azo Compounds/radiation effects , Cell Line, Tumor , Dasatinib/radiation effects , Fusion Proteins, bcr-abl/metabolism , Humans , Lenalidomide/radiation effects , Ligands , Proteolysis/drug effects , Stereoisomerism , Ubiquitin-Protein Ligases , Ubiquitination/drug effects , Ultraviolet RaysABSTRACT
Cyclodextrin glucanotransferase (CGTase) is an important enzyme with multiple functions in starch utilization. In the present study, a fermentation process for the production of CGTase from Escherichia coli harboring the recombinant plasmid pET28b(+)-CGTase was investigated and optimized. The optimal fermentation and expression conditions were 10.0 g/L glycerol, 20.0 g/L tryptone, and 10.0 g/L yeast extract with an initial pH of 7.0, an IPTG concentration of 0.1 mM and an induction temperature of 28 °C for 10 h. The resulting CGTase activity reached up to 36.4 U/L and was 2.1-fold higher than before optimization. Under these optimal fermentation conditions, the up-scaled fermentation was carried out in a 500-L fermentor, and a CGTase activity of 45.2 U/L was achieved. This study provides a foundation for the industrial production of CGTase.
