Fig. 4: Inhibition assays of lead compound 20 against GAR domain-Mrt4 interaction.
From: Inhibiting Mrt4-rRNA interaction with fumaramidmycin-based derivatives as an antifungal strategy
a The cryo-EM structure of the GAR domain-Mrt4 complex in the pre-60S ribosomal particle (PDB 8hfr). b Binding activity of a FAM-labeled ssRNA probe to CaMrt4 with or without 20 treatment, analyzed via Electrophoretic mobility shift assay (EMSA). The final concentrations of 20 in lanes 2–7 were 0, 0.63, 1.25, 2.5, 5, and 10 µM, respectively, while the FAM-labeled ssRNA probe concentration was fixed at 0.1 µM in all the lanes. The final CaMrt4 protein concentration was 5 µM in lanes 2–7, with lane 1 serving as the control (no CaMrt4 added). The RNA probe corresponds to the GAR structural domain of 25S RNA (1217–1286 nt) with 3′-FAM modification. B bound, U unbound. c Half maximal inhibitory concentration (IC50) of 20 for the interaction between the FAM-labeled ssRNA probe and wild-type CaMrt4. d Inhibition rates of cis-fumaramidmycin-based derivatives against the GAR domain-Mrt4 interaction at the concentration of 4 µM. The data are presented as the means ± SDs (n = 3 independent experiments). e–h Binding response curves and dissociation constants (Kd) of the FAM-labeled ssRNA probe with wild-type CaMrt4 cysteine mutants, as measured using FP assays. The protein was pre-incubated with or without 20 (4 µM) overnight at 4 °C. i Overlap of the 3D structures of CaMrt4 and HuMrt4 generated by AlphaFold3, highlighting the cysteine residues in both proteins. j Binding response curves and dissociation constants (Kd) of the FAM-labeled ssRNA probe with HuMrt4 at varying concentrations, with or without pretreatment with 20 (4 µM). All fluorescence polarization assays were performed under identical conditions. The data are presented as the means (n = 2 independent experiments) and the source data are provided as a Source data file.
