PMC

Compound 7. A. Chemical structure. B. X-ray crystal structure. C. ¹H NMR in DMSO-d₆ and D. Mass Spectra.

^aReagents and conditions: (i) AcOH, 3.5-8 h, reflux, 40-58%; (ii) POCl₃, 30-60 min., reflux, 43-65%; (iii) R₂R₃NH, EtOH, 8-15 h, rt, 80-87%.

A. Chemical structure of compound 1. B. Structure of PfDHODH active site. Residues conserved between P. falciparum and human DHODH are displayed in grey, variable residues are displayed in purple. Orotic acid (Oro), FMN and 1 are displayed in yellow. The figure was generated using PyMol from the file 1TV5.pdb.

Selective and potent inhibition of PfDHODH and P. falciparum cells by compound 7. A. Inhibition profile for PfDHODH (orange circles; IC₅₀ 0.047 ± 0.022 μM; n=6) compared to human DHODH (black diamonds; IC₅₀ >200 μM); [E]_T = 10 nM. B. Activity in whole cell assays against P. falciparum 3D7 (orange circles) or mouse L1210 (black diamonds) cells (EC₅₀ = 0.079 ±0.045; n=9). C. Relationship between IC₅₀ and substrate concentration ([E]_T = 5 nM). K_I was determined by fitting the data to (K_I = 0.015 ± 0.0011 μM). D. Rapid kinetic analysis showing compound 7 inhibits the CoQ_D-dependent oxidative half-reaction (bottom panel) but not the DHO-dependent reductive half-reaction (top panel). Blue trace (1) - no compound 7 and orange trace (2) - compound 7 (50 μM).

The biochemical mechanism of inhibition by compound 7 was studied in detail to gain insight into the structural basis for the selective and potent binding of this compound to PfDHODH. Steady-state kinetic analysis shows that the IC₅₀ for compound 7 increases linearly with increasing CoQ_D concentration () as expected for a competitive tight binding inhibitor. These data were fitted to yielding a K_I in the low nanomolar range (K_I = 0.015 ± 0.001 μM). Pre-steady state stopped flow spectroscopy was performed to characterize the effect of compound 7 on the individual oxidative and reductive half-reactions catalyzed by PfDHODH. Compound 7 inhibited the oxidative half reaction (k_ox), preventing the transfer of electrons from FMN to CoQ, while it did not affect the DHO dependent reductive half reaction (k_red) (, ). We previously observed a similar pattern of inhibition for both compound 1 and the biphenylamide inhibitors from our HTS screen. Thus, these data suggest all three inhibitor classes utilize the same mechanism to inhibit DHODH. Finally, site-directed mutagenesis of residues in the inhibitor binding-site (F227A and R265A) of PfDHODH increased the IC₅₀ of compound 7 by 940-fold and 130-fold respectively (IC₅₀ = 44 ± 10 μM for F227A, and IC₅₀ = 6.1 ± 1.1 μM for R265A), providing strong evidence that compound 7 is also bound in this site. As the inhibitor binding-site is not conserved in amino acid composition between P. falciparum and human DHODH this binding mode explains the profound species-selective binding of compound 7 and its derivatives ().