Identification of compounds that inhibit the binding of Keap1a/Keap1b Kelch DGR domain with Nrf2 ETGE/DLG motifs in zebrafish

Abstract

The Keap1‐Nrf2‐ARE system serves as a premier defence mechanism to curb oxidative stress, which remains as one of the major causes of ageing and pathogenesis in various diseases. Nrf2 is the principal master regulator of the cellular defence system, and its activation remains the prospective therapeutic approach against chronic diseases. One of the recent strategies is to disrupt Keap1‐Nrf2 protein‐protein interaction (PPI) that alters the docking of Keap1 with Nrf2 by compounds occupying a position in the Keap1 blocking the interface with Nrf2. In this study, we made an attempt to identify the compounds with anticancer, antioxidant and anti‐inflammatory properties to disrupt Keap1a/b‐Nrf2 PPI through in silico molecular docking in zebrafish. The phylogenetic analysis of Keap1 proteins revealed the existence of orthologous Keap1‐Nrf2‐ARE system in lower vertebrates that includes zebrafish. The DGR domains of zebrafish Keap1a and Keap1b were modelled with Modeller 9.19 using Keap1 of Mus musculus (PDB ID:5CGJ) as template. Based on the docking calculations, top hit compounds were identified to disrupt both Keap1a and Keap1b interaction with Nrf2 which include quercetin 3,4′‐diglucoside, flavin adenine dinucleotide disodium salt hydrate, salvianolic acid A, tunicamycin and esculin. The LC50 of esculin in 3 dpf zebrafish larvae is 5 mmol/L, and the qRT‐PCR results showed that esculin significantly increased the transcription of Nrf2 target genes—Gstpi, Nqo1, Hmox1a and Prdx1 in 3 dpf zebrafish larvae. These potential hits could serve as safer Nrf2 activators due to their non‐covalent disruption of Keap1‐Nrf2 PPI and be developed into efficacious preventive/therapeutic agents for various diseases.

Z-REX uncovers a bifurcation in function of Keap1 paralogs

“…Such adaptability/simplicity is paramount for studying potential off-target proteins identified in large-scale screening assays, or for screening large numbers of compounds that could have numerous on-and off-the-path targets. 21 We also uncovered new insights into AR-regulation orchestrated by the different zKeap1paralogs present in fish. These findings were derived from transient knockdowns, and Z-REX experiments that highlighted differences in responsivity to electrophiles in the head and the tail of the fish.…”

Nrf2/ARE Activators Improve Memory in Aged Mice via Maintaining of Mitochondrial Quality Control of Brain and the Modulation of Gut Microbiome

“…Nfe2l2 contains ARE-like sequences in the promoter region and Nrf2 may activate its own gene expression, leading to increased production of Nrf2 protein [ 29 ]. It is well known that DMF, MB, and RSV activate the Nrf2/ARE signal pathway ( Figure 9 ) [ 30 , 31 , 32 ]. DMF covalently interacts with the reactive cysteines lying in the BTB and IVR regions of Kelch-like ECH-associated protein 1 (Keap1).…”

“…In addition, DMF binds to the Nrf2-binding site (bottom region of Keap1-DC), and to the top region of Keap1-DC, near blade II [ 30 ]. RSV forms H-bonds with Ser49, Asn100, Arg101, Leu196, and Ser288 of the Kelch domain and Pi–cation interaction with Arg101 (motifs in zebrafish) [ 31 ]. RSV also activates Nrf2 through stimulation of the Sirt1/FoxO1 pathways [ 32 ].…”

Validation, Optimization, and Application of the Zebrafish Developmental Toxicity Assay for Pharmaceuticals Under the ICH S5(R3) Guideline

“…The evaluation of malformation and mortality in zebrafish after exposure to compounds was performed following the previous reports from multiple inter-laboratory studies ( Gustafson et al, 2012 ; Ball et al, 2014 ; Raghunath et al, 2019 ). At 4–6 hpf, zebrafish were manually transferred into a 24-well plate (Nest Biotech, Shanghai, China) containing negative compounds, positive compounds, or vehicle (0.5% DMSO), with 12 zebrafish per well in 1 ml of fish water.…”