Abstract by Chunyu Lin
Targeting the Keap1-Nrf2 protein-protein interaction is a potential therapeutic strategy to activate Nrf2 for oxidative stress-related diseases and conditions. It is well-known that identifying protein-protein interaction inhibitors with favorable drug-like properties can be challenging, and in particular for Keap1 due to the highly basic nature of the binding site. Fragment screening combined with structure-based drug design has been suggested as a potential strategy to identify drug-like small-molecule inhibitors.
Here, a complete fragment-based drug discovery (FBDD) study starting with a crystallographic screening of 768 fragments against the mouse Keap1 Kelch domain gave 80 fragment hits binding in the Keap1 Kelch pocket. After analysing the data set and identifying several fragment hits, we tested (after making the hits) a selection of the hits and found that XCF#0252 was active by surface plasmon resonance (SPR) assay (Kd = 0.6 mM). Then, XCF#0252 was selected as a starting point for fragment-to-lead (F2L) elaboration. Fragment-growing guided by crystallography led to a series of naphthalene-based analogues with high selectivity and strong affinity (Ki = 3.5–100 nM). For example, compounds 23n (Ki = 3.5 nM), 23o (Ki = 5.9 nM) and 16n (Ki = 4.7 nM) exhibited a 102,000–171,000-fold increase in affinity compared to the fragment hit XCF#0252. Interestingly, LE rose from the parent fragment XCF#0252 (LE = 0.26) to lead-sized analogues 23n, 23o, and 16n (LE = 0.29–0.30). In the HaCaT cell assay, compounds 23l, 23m, and 16l, demonstrated clear effects on the activation of Keap1-Nrf2 pathway and showed no sign of cytotoxicity. Based on a preliminary analysis of the physicochemical properties and cell assay data, we hypothesize that tPSA < 100 Å2 is a criterion for obtaining membrane penetration and thereby cellular activity for this compound series. We highlight an effective structure-driven F2L elaboration process from the millimolar fragment to nanomolar lead compounds with promising biological activity in cells.