Fig. 5: Triple-site ProRS inhibitors biochemical characterization.
a Interaction of halofuginone with non-hydrolyzable ATP-analog AMP-PNP bound to HsProRS (PDB: 4HVC). Hydrogen-bonds are indicated by yellow dashed lines. b Chemical structures of triple-site inhibitors based on halofuginone-AMP and pyrazinamide-halofuginone hybrid design, with relative stereochemistry shown for halofuginone moiety. c Chemical structures of iso-33 (relative stereochemistry for halofuginone moiety) and cyclization byproduct N3,5’-cycloadenosine. d Dose-response titration of pyrazinamide-halofuginone hybrids using CoraFluor-1-labeled HT-PfcProRS (0.25–0.5 nM) and MAT379 as tracer at 2.5x KD (250 nM). Inhibitors are color coded: 35 (dark blue), 36 (light gray), 37 (dark gray), 38 (blue), 39 (red), MAT436 (gray), and iso-MAT436 (light blue). See Supplementary Fig. 18 for corresponding data in the presence of 100 μM Pro and with HT-HsProRS in the presence or absence of 100 μM Pro. e Chemical structure of iso-MAT436 (40) with relative stereochemistry for halofuginone moiety. f Co-crystal structure of MAT436 (cyan sticks, PDB: 7QC1) bound to PfcProRS reveals electrostatic interactions (dashed lines) with conserved residues within the active site (light blue sticks) and occupancy of all three substrate-binding pockets in the active site. Overlay of co-crystal structures of PfcProRS in complex with MAT436 (cyan sticks, PDB: 7QC1) with either (g) PfcProRS in complex with halofuginone and AMP-PNP (silver sticks, PDB: 4Q15), and (h) PfcProRS in complex with NCP26 (yellow sticks) and proline (orange sticks, PDB: 6T7K). TR-FRET assay data in d are expressed as mean ± s.d. (for 37, 38, 39, and iso-MAT436: n = 2 independent replicate wells; for MAT436, 35, and 36: n = 6) and are representative of at least two independent experiments.
