Structural Characterization of the Covalent Binding of Keap1 and Thiol‐containing Small Molecules

Abstract

Oxidative stress is involved in a wide range of human diseases such as cancer, diabetes, and Alzheimer’s disease. High levels of reactive oxygen species (ROS) cause oxidative damage to DNA and other molecules vital to cell function, such as membrane lipids and proteins. Kelch‐like ECH‐associated protein 1 (Keap1) is a primary regulator of the body’s natural defense system against oxidative stress. In the absence of oxidative stress, Keap1 forms a homodimer that binds a single molecule of transcription factor nuclear factor‐like 2 (Nrf2), causing Nrf2 ubiquitination and degradation. Oxidative stress causes Keap1 to release Nrf2, allowing it to translocate to the nucleus, bind to Maf protein, and activate the production of antioxidant enzymes by binding to the antioxidant response element (ARE). Blocking the protein‐protein interaction (PPI) between Keap1 and Nrf2 could have great therapeutic value. The antioxidant enzymes upregulated by free Nrf2 provide an efficient, catalytic detoxification of ROS. However, as PPIs frequently take place between large, flat interfaces on the two participating proteins, these interactions can be difficult to block. A previous study by our collaborators at UCSF identified 43 thiol‐containing compounds (monophores) that can be covalently tethered to Keap1 at a cysteine near the protein interface. The goal of this project is to characterize the interaction between Keap1 and the monophores using X‐ray crystallography to obtain structures of the protein‐monophore complexes, or adducts. This structural data will allow us to judge which compounds are suitable for scaffold optimization, a technique in which a larger molecule is built from an initial smaller compound to improve its potency and selectivity. Information about the binding modes of the monophores could also be incorporated into the design of other types of drugs that have proven effective in blocking PPIs, such as macrocycles. To identify adduct crystallization conditions, a DTNB assay, which measures the number of reactive sulfhydryl groups in a protein, was used to screen buffer conditions without dithiothreitol (DTT) present. A high‐throughput version of this assay was developed and utilized, in combination with differential scanning fluorimetry (DSF), to identify stabilizing conditions in which adduct can form. This assay was validated with a monophore known to have a stabilizing effect on Keap1, and a new buffer condition (50 mM EPPS, 50 mM NaCl, pH 8.0) was identified in which adduct crystallization attempts can be made.Support or Funding InformationThis work was supported by the Arnold and Mabel Beckman Foundation.