Diverse ligands for mycobacterial CYP124 identified from plant and marine compounds

Tuberculosis persists as a major global health threat, significantly exacerbated by the rise of drug-resistant strains. Cytochrome P450 of 124 family CYP124 from Mycobacterium tuberculosis (CYP124), implicated in host sterol metabolism and bacterial virulence, represents an emerging and promising therapeutic target. While its precise physiological role was previously debated, CYP124's confirmed ability to metabolise immunomodulatory host sterols underscores its pharmacological relevance. Utilizing surface plasmon resonance binding assays and UV-Vis spectral titration screening, we identified nine novel non-azole ligands for CYP124 from a library of 32 plant-derived and marine natural compounds. Among these hits, (25S)-5α-cholestane-3β,4β,6α,7α,8,15β,16β,26-octaol (termed 15β-octaol) and henricioside H₂ (HD-4) induced characteristic difference spectra and formed long-lived inhibitory complexes with CYP124, exhibiting dissociation half-lives of 181 min and 65 min, respectively. However, their inhibitory potency was moderate, with IC₅₀ values of approximately 86 μM for 15β-octaol and exceeding 100 μM for HD-4. Complementary in silico molecular docking and analysis identified key conserved hydrophobic residues within the CYP124 active site crucial for ligand binding, suggesting a shared pharmacophore. Furthermore, structural similarity analysis revealed that 37 human endogenous metabolites, including known immunoregulatory sterols, bear resemblance to the identified CYP124 ligands. This finding points towards a potential sterol-mediated interplay at the host-pathogen interface. Collectively, these results provide a foundation for the future development of mechanism-based CYP124 inhibitors as therapeutics against multidrug-resistant tuberculosis.