scholarly journals Identification of novel inhibitors of Keap1/Nrf2 by a promising method combining protein–protein interaction-oriented library and machine learning

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yugo Shimizu ◽  
Tomoki Yonezawa ◽  
Junichi Sakamoto ◽  
Toshio Furuya ◽  
Masanori Osawa ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Chemical Space ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Aqueous Solubility ◽  
Related Factor ◽  
Binding Modes ◽  
Time Resolved ◽  
Protein Protein Interaction ◽  
Small Molecule Libraries

AbstractProtein–protein interactions (PPIs) are prospective but challenging targets for drug discovery, because screening using traditional small-molecule libraries often fails to identify hits. Recently, we developed a PPI-oriented library comprising 12,593 small-to-medium-sized newly synthesized molecules. This study validates a promising combined method using PPI-oriented library and ligand-based virtual screening (LBVS) to discover novel PPI inhibitory compounds for Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2). We performed LBVS with two random forest models against our PPI library and the following time-resolved fluorescence resonance energy transfer (TR-FRET) assays of 620 compounds identified 15 specific hit compounds. The high hit rates for the entire PPI library (estimated 0.56–1.3%) and the LBVS (maximum 5.4%) compared to a conventional screening library showed the utility of the library and the efficiency of LBVS. All the hit compounds possessed novel structures with Tanimoto similarity ≤ 0.26 to known Keap1/Nrf2 inhibitors and aqueous solubility (AlogP < 5). Reasonable binding modes were predicted using 3D alignment of five hit compounds and a Keap1/Nrf2 peptide crystal structure. Our results represent a new, efficient method combining the PPI library and LBVS to identify novel PPI inhibitory ligands with expanded chemical space.

scholarly journals BRET-based RAS biosensors that show a novel small molecule is an inhibitor of RAS-effector protein-protein interactions

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Nicolas Bery ◽  
Abimael Cruz-Migoni ◽  
Carole JR Bataille ◽  
Camilo E Quevedo ◽  
Hanna Tulmin ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Human Cancers ◽  
Ras Family ◽  
Ras Inhibitors ◽  
In Cells

The RAS family of proteins is amongst the most highly mutated in human cancers and has so far eluded drug therapy. Currently, much effort is being made to discover mutant RAS inhibitors and in vitro screening for RAS-binding drugs must be followed by cell-based assays. Here, we have developed a robust set of bioluminescence resonance energy transfer (BRET)-based RAS biosensors that enable monitoring of RAS-effector interaction inhibition in living cells. These include KRAS, HRAS and NRAS and a variety of different mutations that mirror those found in human cancers with the major RAS effectors such as CRAF, PI3K and RALGDS. We highlighted the utility of these RAS biosensors by showing a RAS-binding compound is a potent pan-RAS-effector interactions inhibitor in cells. The RAS biosensors represent a useful tool to investigate and characterize the potency of anti-RAS inhibitors in cells and more generally any RAS protein-protein interaction (PPI) in cells.

scholarly journals Identification of Small-Molecule Frequent Hitters from AlphaScreen High-Throughput Screens

2013 ◽  
Vol 19 (5) ◽  
pp. 715-726 ◽  
Author(s):  
Kenji Schorpp ◽  
Ina Rothenaigner ◽  
Elena Salmina ◽  
Jeanette Reinshagen ◽  
Terence Low ◽  
...  
Keyword(s):  
Small Molecule ◽  
Detection System ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Assays ◽  
Time Resolved ◽  
Protein Protein Interaction ◽  
Nickel Chelate ◽  
High Throughput Screens

Although small-molecule drug discovery efforts have focused largely on enzyme, receptor, and ion-channel targets, there has been an increase in such activities to search for protein-protein interaction (PPI) disruptors by applying high-throughout screening (HTS)–compatible protein-binding assays. However, a disadvantage of these assays is that many primary hits are frequent hitters regardless of the PPI being investigated. We have used the AlphaScreen technology to screen four different robust PPI assays each against 25,000 compounds. These activities led to the identification of 137 compounds that demonstrated repeated activity in all PPI assays. These compounds were subsequently evaluated in two AlphaScreen counter assays, leading to classification of compounds that either interfered with the AlphaScreen chemistry (60 compounds) or prevented the binding of the protein His-tag moiety to nickel chelate (Ni2+-NTA) beads of the AlphaScreen detection system (77 compounds). To further triage the 137 frequent hitters, we subsequently confirmed by a time-resolved fluorescence resonance energy transfer assay that most of these compounds were only frequent hitters in AlphaScreen assays. A chemoinformatics analysis of the apparent hits provided details of the compounds that can be flagged as frequent hitters of the AlphaScreen technology, and these data have broad applicability for users of these detection technologies.

scholarly journals Bioluminescence Resonance Energy Transfer as a Method to Study Protein-Protein Interactions: Application to G Protein Coupled Receptor Biology

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 537 ◽  
Author(s):  
Chayma El Khamlichi ◽  
Flora Reverchon-Assadi ◽  
Nadège Hervouet-Coste ◽  
Lauren Blot ◽  
Eric Reiter ◽  
...  
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Receptor Biology ◽  
G Protein Coupled

The bioluminescence resonance energy transfer (BRET) approach involves resonance energy transfer between a light-emitting enzyme and fluorescent acceptors. The major advantage of this technique over biochemical methods is that protein-protein interactions (PPI) can be monitored without disrupting the natural environment, frequently altered by detergents and membrane preparations. Thus, it is considered as one of the most versatile technique for studying molecular interactions in living cells at “physiological” expression levels. BRET analysis has been applied to study many transmembrane receptor classes including G-protein coupled receptors (GPCR). It is well established that these receptors may function as dimeric/oligomeric forms and interact with multiple effectors to transduce the signal. Therefore, they are considered as attractive targets to identify PPI modulators. In this review, we present an overview of the different BRET systems developed up to now and their relevance to identify inhibitors/modulators of protein–protein interaction. Then, we introduce the different classes of agents that have been recently developed to target PPI, and provide some examples illustrating the use of BRET-based assays to identify and characterize innovative PPI modulators in the field of GPCRs biology. Finally, we discuss the main advantages and the limits of BRET approach to characterize PPI modulators.

Development of a Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay for the Inhibition of Keap1–Nrf2 Protein–Protein Interaction

2020 ◽  
Vol 26 (1) ◽  
pp. 100-112
Author(s):  
Sumi Lee ◽  
Dhulfiqar Ali Abed ◽  
Lesa J. Beamer ◽  
Longqin Hu
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Small Molecule ◽  
Protein Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Protein Protein Interaction ◽  
Nrf2 Protein

The transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), plays a major role in regulating the antioxidant defense system through the Kelch-like ECH-associated protein 1–Nrf2–antioxidant response element (Keap1–Nrf2–ARE) pathway. Small-molecule inhibitors targeting Keap1–Nrf2 protein–protein interaction (PPI) decrease the rate of Nrf2 degradation by the 26S proteasome and thus increase the intracellular level of Nrf2, which translocates into the nucleus, leading to upregulated expression of cytoprotective and antioxidant enzymes. Such inhibitors can be developed into potential preventive and therapeutic agents of diseases caused by oxidative damage. To more effectively identify promising Nrf2 activators through the inhibition of Keap1–Nrf2 PPI, a homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assay was developed in this work by indirectly labeling the Keap1 Kelch domain protein with Tb-anti-His antibody as the donor and using, as the acceptor, fluorescein isothiocyanate (FITC)-labeled 9mer Nrf2 peptide amide, the same fluorescent probe that was used in an earlier fluorescence polarization (FP) assay. Assay conditions, including concentrations of the various components, buffer type, and incubation time, were optimized in the TR-FRET competition assay with known small-molecule inhibitors of Keap1–Nrf2 PPI. Under the optimized conditions, the Keap1–Nrf2 TR-FRET assay exhibited great sensitivity with a high dynamic range and considerable stability for as long as 5 h. The Z’ factor was determined to be 0.82, suggesting that the assay is suitable for high-throughput screening and lead optimization of inhibitors of Keap1–Nrf2 PPI. Furthermore, the TR-FRET assay is capable of differentiating potent inhibitors of Keap1–Nrf2 PPI down to the subnanomolar inhibition constant ( Ki) range.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

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