Breaking down Keap1 ligands into fragments enables scientists to (re)construct new potent compounds

Inhibition of the interaction between Keap1 and Nrf2 proteins can help to prevent and reduce oxidative stress which, when left untreated, can cause neurological diseases, cancer, as well as cell and tissue damage. In a new project, scientists broke down existing compounds that have shown the ability to inhibit the Keap1–Nrf2 protein-protein interaction into fragments and assessed if these smaller versions of the compounds were able to bind Keap1. X-ray crystallography, assisted by MAX IV’s BioMAX beamline, showed that the approach referred to as ‘fragment-based deconstruction reconstruction’ yielded 17 ‘fragment hits’ of low weight molecules with six shown to bind the target protein Keap1’s Kelch binding domain. These insights confirm FBDR as an effective method for finding new ’fragment hits,’ for understanding ligand–protein interactions, and for identification of new molecules that decrease the activity of Keap1–Nrf2 protein-protein interaction.

‘Fragment hits’ describe molecules of low molecular weight that demonstrably bind to a target protein and can therefore serve as ideal entry points for new drug development. Keap1 is a protein coding gene which encodes kelch-like ECH-associated protein 1 in humans. It interacts with nuclear factor erythroid 2-related factor 2 (Nrf2), a protein that controls the rate of transcription of genetic information from DNA to messenger RNA in humans. When Nrf2 becomes active, it generates an antioxidant and anti-inflammatory response in the human body. Researchers have found that Keap1 represses Nrf2 activation and thus increases the amount of free radicals and chain reactions. This discovery has turned Keap1 into a very attractive target for drug development. Targeting the protein–protein interaction (PPI) between the Neh2 domain of Nrf2 and the Kelch domain of Keap1 has been proposed as a potential therapeutic strategy to lessen oxidative stress.

Structure of the KEAP1 protein. Based on PyMOL rendering of PDB 1u6d.
Attribution: Emw, CC BY-SA 3.0, via Wikimedia Commons

”In order to make new molecules that bind the Keap1 kelch domain we use a technique called fragment-based drug discovery (FBDD). The principle is to screen for small substructures of typical drug-like molecules, so-called fragments, and then optimize the fragment hits into larger and more potent lead molecules. This is in contrast to traditional high-throughput screenings, where one directly screens for larger molecules with an expected stronger binding to the protein target,“ explained University of Copenhagen Associate Professor for Medicinal Chemistry Anders Bach.

For this reason, Bach together with fellow scientists from the University of Copenhagen, the Miami Miller School of Medicine, and the Technical University of Munich employed an alternative method that expands on the possibilities offered by FBDD by dissecting inhibitors and creating a target-biased library of fragments. This means that rather than screening for standard or general fragment hits, they created their own fragment library predisposed for the target in mind. This resulted in a high hit rate and good fragment hits that could be optimized to nanomolar potent molecules. This strategy is called fragment-based deconstruction reconstruction (FBDR).

Assisted through beamlines including MAX IV’s BioMAX and X-ray crystallography, the researchers were able to employ FBDD to confirm that the fragment hits identified by biophysical methods actually bind the target (Keap1), and to find out exactly where on the protein the hits bind. They compared six fragment hits binding the part of Keap1 referred to as ’Kelch’ domain with the binding modes of the original compounds allowing them to highlight several hot spot anchor points with enhanced binding affinity. This domain is a well-defined entity of the Keap1 protein, shaped like a bowl which forms a relatively large protein pocket which Nrf2 fits into. The Keap1 Kelch domain has previously been divided into five subpockets (P1-5), which bind small-molecules and Nrf2. These subpocket designations serve the description and discussion of the molecular interactions between Keap1 and ligands. Thanks to this structural insight they selected promising fragment hits and merged them into new compounds with uniquely pronounced affinities.

“FBDD studies of Keap1 have used generic fragment libraries to find new fragment hits. What we did was slightly different: We generated our own fragment-library by dissecting already existing small-molecule Keap1 inhibitors into a deconstruction library of 77 fragments,” said Bach.  “With such a target-biased library we expected to get a high hit-rate; and the fragment-hits would at the same time correspond to energetically favorable parts of the bigger molecules,” said Bach.

The X-ray structures obtained through this study provided new insights into the molecular conformations of the ligand and the protein-ligand interactions that lead to high affinity. To the researchers’ surprise, affinity gain could be obtained via the upper parts of P1, and not the lower parts as expected based on prior computational analyses – an insight that can be useful for the future design of Keap1 inhibitors.

This information was essential for the subsequent chemical optimization phase and combination of promising structural features, which resulted in the creation of additional compounds with high binding affinities. All of the newly created compounds proved to be more resistant to microsomal degradation than their original versions.

These results promote FBDR as an effective method at the forefront for finding new ‘fragment hits’ and for building new potent Keap1–Nrf2 PPI inhibitors. Bach and his colleagues expect that FBDR may serve as a powerful strategy for other challenging drug targets as well: ”With the many X-ray structures included in this study, we provide a unique level of insight into the structural requirements for small-molecules binding to Keap1. Overall, the technical advances, fragment hits, lead compounds, and structural knowledge presented here can guide future drug discovery.”


Pallesen, J. S., Narayanan, D., Tran, K. T., et al. Deconstructing Noncovalent Kelch-like ECH-Associated Protein 1 (Keap1) Inhibitors into Fragments to Reconstruct New Potent Compounds. Journal of medicinal chemistry, 64 (8), 4623–4661. (2021), DOI:

Cover image attribution: ‘Protein-Ligand Interaction Simulations and Analysis’ by T. Andrew Binkowski, Ph.D., Midwest Center for Structural Genomics & Biosciences Division, Argonne National Laboratory; Center for Structural Genomics of Infectious Diseases & Computation Institute, The University of Chicago via Flickr