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  • Some pyrimidine analogs are substrate based inhibitors that


    Some pyrimidine analogs are substrate-based inhibitors that bind to the dihydroorotate binding site, but most reported inhibitors of DHODH bind to the site occupied by the ubiquinone co-factor., , , , , , , , , , , , , , , , , X-ray crystallographic studies of inhibitor complexes with DHODH and DHODH have revealed that known inhibitors, such as brequinar and teriflunomide (the active metabolite of leflunomide), inhibit enzyme activity by displacing the ubiquinone co-factor,, , , , , , either competitively or otherwise. Analogs of brequinar and teriflunomide all contain an acidic head group that interacts with the guanidinyl group of Arg-136 of DHODH, or the corresponding Arg-265 of DHODH, at the ubiquinone binding site. An alternative binding mode wherein the acidic group interacts with Tyr-356 has been reported, with some compounds adopting a dual binding mode. This dual binding mode has been observed in a species dependent manner, where the hydroxyl group of teriflunomide interacts with Arg-136 in DHODH but with Tyr-528 in DHODH. Compounds with a neutral polar head group have also been reported as DHODH inhibitors,, including the recently disclosed triazolopyrimidine derivatives, and S-2678. The X-ray structures showed that the triazolopyrimidine head group acts as a hydrogen-bond acceptor, interacting with the guanidinyl group of Arg-265 in the DHODH enzyme. In a search for novel inhibitors of human DHODH, we carried out virtual screening of the sanofi-aventis Ac-Endothelin-1 (16-21), human mg collection and subsequently tested the selected compounds in a human DHODH enzymatic assay. We also determined the binding mode of selected hits by X-ray crystallography. We report here several amino-benzoic acids as novel inhibitors of DHODH discovered by virtual screening and confirmed by experimental studies. The software program GLIDE was used for virtual screening with the initial vHTS setting followed by the SP procedure. Virtual screening was carried out by molecular docking (PDB: ) with constraints of H-bonding interaction to the side-chain of either Arg-136 or Tyr-365 and a hydrophobic pharmacophore feature 6–8Å away from the hydrogen-bond. Selected compounds were tested in the human DHODH enzymatic assay, which resulted in 200 hits with IC <10μM (in comparison to an IC of 3nM for brequinar). Four of these hits are shown in . 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 acid 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.