Tetrazole-containing naphthalene bis-sulfonamide Keap1-Nrf2 interaction inhibitors with unexpected binding modes.

Naphthalene bis-sulfonamides are amongst the most studied inhibitors of the interaction between Kelch-like ECH-associated protein-1 (Keap1), a ubiquitination facilitator protein and the nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor associated with diverse cytoprotective responses. The compounds bind to the C-terminal Kelch domain of Keap1 and occupy the Nrf2 binding pocket, causing Nrf2 to accumulate and increase the expression of a battery of antioxidant response element genes. The binding modes of the compounds have been characterised through a series of X-ray crystallography and binding assay studies. In this work we have evaluated a series of symmetric and asymmetric naphthalene bis-sulfonamides and identified a tight binding acid/tetrazole derivative 25. We have determined its distinct binding mode to Keap1 using X-ray crystallography, biophysical binding assays and its Nrf2 activation using cell-based assays. We have also characterised its conformational interconversions in solution using NMR. In contrast to previous studies, we have shown that the compound can bind to Keap1 in both its 'cis' and 'trans' rotational states that are both occupied and interconvert in solution. Molecular dynamics simulations indicate that the cis-form has the lowest interaction energy, but the trans-form is more stable in solution. The compound binds tightly to Keap1 (low nanomolar Kd), indicating that the non-standard binding modes may contribute significantly to the high affinity. The study highlights the need for caution when interpreting the structure-activity relationships of closely related compounds in this series and will inform further work on these compounds.