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The Keap1-Nrf2 system is an evolutionarily conserved defense mechanism against oxidative and xenobiotic stress. Its regulatory mechanisms, e.g., stress-sensing mechanism, proteasome-based regulation of Nrf2 activity and selection of target genes, have been elucidated mainly in mammals. In addition, emerging model animals, such as zebrafish, fruit fly and Caenorhabditis elegans, have been shown to have similar anti-stress systems to mammals, suggesting that analogous defense systems are widely conserved throughout the animal kingdom. Experimental evidence in lower animals provides important information beyond mere laboratory-confined utility, such as regarding how these systems transformed during evolution, which may help characterize the mammalian system in greater detail. Recent advances in genome projects of both model and non-model animals have provided a great deal of useful information toward this end. We herein review the research on Keap1-Nrf2 and its analogous systems in both mammals and lower model animals. In addition, by comparing the amino acid sequences of Nrf2 and Keap1 proteins from various species, we can deduce the evolutionary history of the anti-stress system. This combinatorial approach using both experimental and genetic data will suggest perspectives of approach for researchers studying the stress response.

Vascular calcification is a complication of diseases and conditions such as chronic kidney disease, diabetes, and aging. Previous studies have demonstrated that high concentrations of inorganic phosphate (Pi) can induce oxidative stress and vascular smooth muscle cell calcification. KEAP1 (Kelch-like ECH-associated protein 1)/NF-E2-related factor 2 (NRF2) signaling has been shown to play important roles in protecting cells from oxidative stress. The current study aims to investigate the possible involvement of the KEAP1/NRF2/P62 -mediated antioxidant pathway in vascular calcification induced by high Pi levels. Exposure of vascular smooth muscle cells (VSMCs) to high Pi concentrations promoted the accumulation of reactive oxygen species (ROS) and the nuclear translocation of NRF2, along with an increase in P62 levels and a decrease in KEAP1 levels. A classic NRF2 activator, tert-butylhydroquinone (tBHQ), significantly decreased ROS levels and calcium deposition in VSMCs by promoting the nuclear translocation of NRF2 and upregulating P62 and KEAP1 expression. In contrast, silencing NRF2 and P62 with siRNAs increased the levels of ROS and calcium deposition in VSMCs. In conclusion, VSMC calcification can be alleviated by the activation of the KEAP1/NRF2/P62 antioxidative pathway, which could have a protective role when it is exogenously activated by tBHQ.

The transcription factor NF-E2 p45-related factor 2 (NRF2; encoded by NFE2L2) and its principal negative regulator, the E3 ligase adaptor Kelch-like ECH-associated protein 1 (KEAP1), are critical in the maintenance of redox, metabolic and protein homeostasis, as well as the regulation of inflammation. Thus, NRF2 activation provides cytoprotection against numerous pathologies including chronic diseases of the lung and liver; autoimmune, neurodegenerative and metabolic disorders; and cancer initiation. One NRF2 activator has received clinical approval and several electrophilic modifiers of the cysteine-based sensor KEAP1 and inhibitors of its interaction with NRF2 are now in clinical development. However, challenges regarding target specificity, pharmacodynamic properties, efficacy and safety remain.

Inflammation is a key driver in many pathological conditions such as allergy, cancer, Alzheimer’s disease, and many others, and the current state of available drugs prompted researchers to explore new therapeutic targets. In this context, accumulating evidence indicates that the transcription factor Nrf2 plays a pivotal role controlling the expression of antioxidant genes that ultimately exert anti-inflammatory functions. Nrf2 and its principal negative regulator, the E3 ligase adaptor Kelch-like ECH- associated protein 1 (Keap1), play a central role in the maintenance of intracellular redox homeostasis and regulation of inflammation. Interestingly, Nrf2 is proved to contribute to the regulation of the heme oxygenase-1 (HO-1) axis, which is a potent anti-inflammatory target. Recent studies showed a connection between the Nrf2/antioxidant response element (ARE) system and the expression of inflammatory mediators, NF-κB pathway and macrophage metabolism. This suggests a new strategy for designing chemical agents as modulators of Nrf2 dependent pathways to target the immune response. Therefore, the present review will examine the relationship between Nrf2 signaling and the inflammation as well as possible approaches for the therapeutic modulation of this pathway.

On average, an approved drug currently costs US$2–3 billion and takes more than 10 years to develop 1 . In part, this is due to expensive and time-consuming wet-laboratory experiments, poor initial hit compounds and the high attrition rates in the (pre-)clinical phases. Structure-based virtual screening has the potential to mitigate these problems. With structure-based virtual screening, the quality of the hits improves with the number of compounds screened 2 . However, despite the fact that large databases of compounds exist, the ability to carry out large-scale structure-based virtual screening on computer clusters in an accessible, efficient and flexible manner has remained difficult. Here we describe VirtualFlow, a highly automated and versatile open-source platform with perfect scaling behaviour that is able to prepare and efficiently screen ultra-large libraries of compounds. VirtualFlow is able to use a variety of the most powerful docking programs. Using VirtualFlow, we prepared one of the largest and freely available ready-to-dock ligand libraries, with more than 1.4 billion commercially available molecules. To demonstrate the power of VirtualFlow, we screened more than 1 billion compounds and identified a set of structurally diverse molecules that bind to KEAP1 with submicromolar affinity. One of the lead inhibitors (iKeap1) engages KEAP1 with nanomolar affinity (dissociation constant ( K d ) = 114 nM) and disrupts the interaction between KEAP1 and the transcription factor NRF2. This illustrates the potential of VirtualFlow to access vast regions of the chemical space and identify molecules that bind with high affinity to target proteins. VirtualFlow, an open-source drug discovery platform, enables the efficient preparation and virtual screening of ultra-large ligand libraries to identify molecules that bind with high affinity to target proteins.

Several indole derivatives have been disclosed by our research groups that have been collaborating for nearly 25 years. The results of our investigations led to a variety of molecules binding selectively to different pharmacological targets, specifically the type A γ-aminobutyric acid (GABAA) chloride channel, the translocator protein (TSPO), the murine double minute 2 (MDM2) protein, the A2B adenosine receptor (A2B AR) and the Kelch-like ECH-associated protein 1 (Keap1). Herein, we describe how these works were conceived and carried out thanks to the versatility of indole nucleus to be exploited in the design and synthesis of drug-like molecules.

Background Kelch‐like ECH‐associated protein 1 (KEAP1) has been identified as a cancer driver gene in lung adenocarcinoma (LUAD), and increased evidence has given us clues about the association of KEAP1 mutation and immune characteristics. We assessed the association between KEAP1 mutation and tumor microenvironment in LUAD systematically. Methods With the data collected from The Cancer Genome Atlas (TCGA), we evaluated the association of KEAP1 mutation with tumor infiltrating leukocytes (TILs), including dendritic cell, CD8 T cell, CD4 T cell, neutrophil, B cells, and macrophage. Expression differences of the markers of those immune cells were also measured. We further compared the expression of antigen presentation genes and chemokines and the enrichment score of immune‐related pathways. Results KEAP1 mutation had significant association with lower TILs and cytotoxic T lymphocyte. Strikingly, almost all of antigen presentation genes and chemokine showed lower expression in KEAP1‐mutated tumors. Moreover, most of immune‐related pathways were less active in KEAP1‐mutated tumors. As expected, KEAP1‐wild type LUADs favored better overall survival after immunotherapy. Finally, one patient harboring KEAP1 mutation along with a lack of immune cells infiltration in tumor microenvironment failed to respond to checkpoint inhibitor despite high tumor mutational burden (TMB). Conclusions KEAP1 mutation has a significant effect on the tumor immune milieu of LUAD and may play as a predictive biomarker of immunotherapy for LUAD patients. Immune cells, the key markers of immune cells, and HLA molecules show significantly down‐expressed in KEAP1‐mutated LUADs. Not only the multiple immune‐related biological processes, but also the interaction between chemokines and their receptors have shown KEAP1 mutation could impair the immune pathways. KEAP1‐wild type LUADs favored better overall survival after immunotherapy.

Oxidative stress can affect the protein, lipids, and DNA of the cells and thus, play a crucial role in several pathophysiological conditions. It has already been established that oxidative stress has a close association with inflammation via nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway. Amino acids are notably the building block of proteins and constitute the major class of nitrogen-containing natural products of medicinal importance. They exhibit a broad spectrum of biological activities, including the ability to activate NRF2, a transcription factor that regulates endogenous antioxidant responses. Moreover, amino acids may act as synergistic antioxidants as part of our dietary supplementations. This has aroused research interest in the NRF2-inducing activity of amino acids. Interestingly, amino acids' activation of NRF2-Kelch-like ECH-associated protein 1 (KEAP1) signaling pathway exerts therapeutic effects in several diseases. Therefore, the present review will discuss the relationship between different amino acids and activation of NRF2–KEAP1 signaling pathway pinning their anti-inflammatory and antioxidant properties. We also discussed amino acids formulations and their applications as therapeutics. This will broaden the prospect of the therapeutic applications of amino acids in a myriad of inflammation and oxidative stress-related diseases. This will provide an insight for designing and developing new chemical entities as NRF2 activators.

Small cyclic peptides have gained significant traction as a therapeutic modality; however, the development of deep learning methods for accurately designing such peptides has been slow, mostly due to the lack of sufficiently large training sets. Here, we introduce AfCycDesign, a deep learning approach for accurate structure prediction, sequence redesign, and de novo hallucination of cyclic peptides. Using AfCycDesign, we identified over 10,000 structurally-diverse designs predicted to fold into the designed structures with high confidence. X-ray crystal structures for eight tested de novo designed sequences match very closely with the design models (RMSD < 1.0 Å), highlighting the atomic level accuracy in our approach. Further, we used the set of hallucinated peptides as starting scaffolds to design binders with nanomolar IC 50 against MDM2 and Keap1. The computational methods and scaffolds developed here provide the basis for the custom design of peptides for diverse protein targets and therapeutic applications. AfCycDesign: Cyclic offset to the relative positional encoding in AlphaFold2 enables accurate structure prediction, sequence redesign, and de novo hallucination of cyclic peptide monomers and binders.

Most quality control pathways target misfolded proteins to prevent toxic aggregation and neurodegeneration 1 . Dimerization quality control further improves proteostasis by eliminating complexes of aberrant composition 2 , but how it detects incorrect subunits remains unknown. Here we provide structural insight into target selection by SCF–FBXL17, a dimerization-quality-control E3 ligase that ubiquitylates and helps to degrade inactive heterodimers of BTB proteins while sparing functional homodimers. We find that SCF–FBXL17 disrupts aberrant BTB dimers that fail to stabilize an intermolecular β-sheet around a highly divergent β-strand of the BTB domain. Complex dissociation allows SCF–FBXL17 to wrap around a single BTB domain, resulting in robust ubiquitylation. SCF–FBXL17 therefore probes both shape and complementarity of BTB domains, a mechanism that is well suited to establish quality control of complex composition for recurrent interaction modules. Structural studies of the dimerization quality control E3 ubiquitin ligase SCF–FBXL17 indicate that its selectivity for aberrant complex formation is based on recognizing both shape and complementarity of interacting domains.