The binding modes of the compounds

The binding modes of the compounds were revealed by co-crystal structures with DHODH, determined by X-ray crystallography. The compounds are found at the coenzyme Q site, as expected. -dihydroorotate (DHO), and flavin mononucleotide (FMN) are also observed in the structures which were solved at a resolution of 1.9–2.1Å. The overall position and the orientation of the Chrysin synthesis group toward Arg-136 is similar to that previously observed for brequinar in human DHODH (1D3G). For compound (shown in ), a hydrogen-bond network is observed around the acidic group that makes bifurcated hydrogen-bonds to the guanidinyl group of Arg-136. One carboxylate oxygen atom forms a hydrogen-bond to a crystallographic water molecule, which hydrogen-bonds to Gln-47. The other carboxylate oxygen hydrogen bonds to a second crystallographic water, which in turn hydrogen-bonds to the NHs of the urea linker of the inhibitor and the backbone carbonyl oxygen of Thr-360. The benzoic acid phenyl ring lies in a hydrophobic pocket formed by the flavin mononucleotide and the side-chains of Val-134 and Tyr-356, limiting potential substitutions on the ring that could increase potency. In contrast, the dichlorophenyl ring points toward the aqueous phase () and offers several opportunities for structural modification to improve the physical properties of the compounds. A similar hydrogen-bond network is observed around the acidic group of inhibitor (); the crystallographic water is in a position nearly identical to that observed with compound , being hydrogen-bonded to the benzoic -NH. Compared with (in purple), inhibitor (in yellow) extends further toward the solvent, as a result of the interposition of a furan ring. The aromatic chlorophenyl group may be favored in this position, due to its proximity to the side-chains of Leu-42, Tyr-38, Leu-68 (hidden behind inhibitors in ), and Phe-62, which form a hydrophobic pocket near the entrance to the ligand binding site. The central region of the inhibitor and the furan ring do not form any close-contact interactions with the protein. The chlorine of the phenyl ring is buried and is 3.0Å from the side-chain hydroxyl of Thr-63. The carbon of the chlorophenyl ring to the furan is fully exposed to the solvent.Compounds and were selected by virtual screening. When tested, both compounds showed good activity (ICs of 5.5 and 0.1μM, respectively). However, when the amides and were re-synthesized, they showed weak inhibition in the DHODH enzymatic assay, suggesting that the activities of the initial samples may be attributed to impurities or hydrolyzed products. Indeed, the -alkyl group is not seen in the original crystallographic electron density omit map prior to positioning either inhibitor in DHODH, nor can it be found in the final maps. Rather, the electron density maps are consistent with hydrolysis of the amide to the acid. Once the scaffold was clearly defined, compound was fitted within the electron density, leaving no space for the -alkyl group. The final electron density map is fully consistent with the structure of (shown in a), including the conformations around the ethyl-amide and the N-linked methylpyridine.