Fig. 4: Detection of CYP inhibition after azamulin (AZA) treatment.

a Schematic diagram of a possible mechanism of AZA binding with CYP. Active CYPs maintain a ferric low-spin (LS) state by using water as a ligand. After AZA enters the enzymatic pocket, water is replaced by AZA, which causes LS to high-spin (HS) transition. Although metabolic intermediates of AZA were not identified, the crystal structure indicated the pleuromutilin head is required for mechanism-based inhibition. The possible ligation was depicted as a black dotted line. b The Raman shifts at 1370 cm⁻¹ and 1636 cm⁻¹ decreased after treatment with AZA. c Comparison of average Raman signal at 1636 cm⁻¹ and CYP3A4 activity assay. Error bars indicate SD between triplicates. ***P < 0.001; **P < 0.01. d Reconstructed Raman images at 600 cm⁻¹, 675 cm⁻¹, 1370 cm⁻¹, and 1636 cm⁻¹. Scale bars, 20 µm. e Immunostaining of cyt b₅ and CYP3A4 at the same positions of Raman measurement. Scale bars, 20 µm.