Novel Biology and Druggable Targets via Chemoproteomics

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Every biochemistry textbook begins by detailing the molecular building blocks of life such as sugars, amino acids, and nucleosides. These overviews begin by illustrating the similar properties between the molecules followed by differences. For instance, proteogenic alpha amino acids share an amine, carboxylate, and chiral alpha carbon, but differ in the chemical properties of their side chains. Of the natural, common amino acids, cysteine has always filled a distinct niche because it possesses a thiol. Through this sulfur atom, cysteine serves multiple functions in biochemistry. In cysteine proteases, for example, cysteine serves as the nucleophile needed to attack and cleave thioester and amide bonds. Other functions for cysteine include metal chelation, structure formation and stabilization through formation of disulfide bonds, and a site of post-translational modifications (i.e. redox chemistry, palmitoylation).

Cysteine is an indispensable amino acid in biology and biochemistry and measuring changes in cysteine reactivity¹ in a proteome-wide manner is now feasible through innovative chemoproteomics methods. These studies can identify and characterize cysteines involved in enzyme catalysis or cysteines that are post-translationally regulated. Isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP) is a cysteine reactivity profiling approach pioneered by the Cravatt lab¹. This method uses an iodoacetamide alkyne as a chemoproteomics probe to label cysteines.

In an impressive new study², Bar-Peled, Cravatt, and coworkers use isoTOP-ABPP to determine proteins and cysteines on those proteins regulated by the nuclear factor E2-related factor 2 (NRF2) transcription factor, the master regulator of cellular antioxidant response pathway³ to identify druggable targets in non-small-cell lung cancers. Non-small-cell lung cancers often have active NRF2⁴. NRF2 activation protects cells from oxidative stress, and inhibition of the NRF2 pathway leads to cell death, but no pharmacological inhibitors of NRF2 are available.

Bar-Peled and colleagues hypothesized that a clearer understanding of oxidative changes controlled by NRF2 would reveal drug targets in this pathway. They used IsoTOP-ABPP to analyze differences in cysteine reactivity in human non-small-cell lung cancers cell lines with mutations in the negative regulator of NRF2, Kelch-like ECH-associated protein 1 (KEAP1). KEAP1 binds to and promotes NRF2 degradation. Analysis of KEAP1 mutant cell lines with overactive NRF2, isoTOP-ABPP revealed NRF2-regulated proteins, which included proteins with jobs in the antioxidant response and metabolism of glutathione, heme, and NADPH.

To determine which of the NRF2-regulated proteins are druggable, they performed a competitive labeling study. In this experiment, Bar-Peled and coworkers measured the capability of two small molecule chloroacetamides to label cysteines on NRF2-regulated proteins and, prevents iodoacetamide alkyne labeling. Chloroacetamide labeling of a cysteine requires binding of these small molecules to a binding pocket near the cysteine to revealing druggable proteins (i.e. proteins with a small-molecule binding pocket). Three of the chloroacetamide-sensitive proteins—NR0B1, CYP4F11, and AKR1B10—were selected for further investigation because they were only expressed in cells with active NRF2.

Tests of NR0B1, CYP4F11, and AKR1B10 for their function in an anchorage-independent growth assay using shRNA knockdown, and all of them were needed for successful cell growth under the assay conditions. Of these proteins, the orphan nuclear receptor NR0B1 was interesting because its restricted tissue expression meant that it might be specific for lung cancers and, therefore, an optimal target.

Structural studies with NR0B1 show that this nuclear receptor lacks a traditional ligand-binding pocket found on most nuclear receptors⁵, but the potential selectivity for cancer afforded by this gene warranted additional mechanistic studies. Integration of gene expression analysis with protein-protein interaction experiments revealed that NR0B1 interacts with SNW domain-containing protein 1 (SNW1), a transcriptional regulator, and RNA-binding protein 45 (RBM45). Cysteine 274 (C274) of NR0B1 (Fig. 1), the iodoacetamide alkyne site of labeling, is in a region of NR0B1that interacts with SNW1. A better ligand for C274 of NR0B1 might disrupt the NR0B1-SNW1 protein interaction to inhibit the NFR2 pathway.

Figure. 1.

NR0B1 is a druggable target downstream of NRF2 that regulates the biological function of the NRF2 pathway. The identification of NR0B1 and the demonstration that inhibition regulates anchorage independent growth demonstrates that the chemical proteomics methods used can reveal new targets for pathways where the known biology offers no feasible pharmacological targets. In this case, the NRF2 pathway is activated in many non-small cell lung cancers (NSCLCs) but this approach should be applicable to other pathways as well.

Screens for electrophilic compounds that inhibited NR0B1 protein-protein interactions identified two drug-like chemicals BPK-26 and BPK-29 that abrogated the interaction between NR0B1 and a critical binding partner SNW1. Tests of BPK-29, along with 2 inactive compounds revealed the specific contribution of chemical inhibition of NR0B1. BPK-29, but not control compounds, inhibits anchorage-independent growth and that this growth inhibition (Fig. 1). As a result, BPK-29 is a small-molecule inhibitor of the NRF2 pathway, providing the first pharmacological inhibitor of this pathway.

Bar-Peled’s remarkable work attained the lofty goal of identifying a new target of the NRF2 pathway, the nuclear receptor NR0B1, utilizing the chemoproteomics of cysteine. Furthermore, having identified that the labeled cysteine residue in NR0B1, Cys274 is at a protein-protein interaction interface critical for NR0B1 function they developed selective inhibitors that inhibit NR0B1 activity. In doing so, they took an undruggable pathway and developed a pharmacological agent capable of targeting this pathway (Fig. 1). Medicinal chemistry and pharmacology can build off these lead compounds to develop therapeutics for NSCLC. Furthermore, the unique platform developed by Bar-Peled and colleagues can be applied to additional pathways that currently lack pharmacological targets.