Figure 1. The ApaG Fbxo3 domain serves as a target for small molecules.

A. Several deletion mutants of Fbxo3 were designed and cloned into a pcDNA3.1D/V5-HIS vector. B. In vitro ubiquitination assays. Purified SCF^Fbxo3 full-length (FL) or truncated Fbxo3 proteins were incubated with V5-Fbxl2 substrate and the full complement of ubiquitination reaction components showing polyubiquitinated Fbxl2 (second lane from left). C. Structural analysis of the Fbxo3-ApaG domain showing highly conserved bacterial (left) and mammalian Fbxo3 (second from left) structures. D. Structure of the base compound, benzathine, and the Fbxo3 antagonist, BC-1215. Right graphs. Human peripheral blood mononuclear (PBMC) cells were treated with LPS (2 µg/ml) for 16 h along with either benzathine or BC-1215 at different concentrations and IL1β and TNFα monitored to calculate the IC₅₀. For LD₅₀, U937 monocytes were treated with the small molecules at different concentrations for 16 h. Cells were then stained with trypan blue to identify dead cells, and to calculate the LD₅₀. E–F. Docking studies of the base compound, benzathine, interacting with the Fbxo3-ApaG domain. Data from panel B represent n=2 separate experiments.