This hydrophobic biphenyl tail gave good binding
This hydrophobic biphenyl tail gave good binding affinity for human DHODH enzyme (IC <0.2μM). As shown in , several compounds—for example, the 3-chloro (, 31nM) and 3-trifluoromethoxy (; 27nM) and closely related analogs—were particularly potent. Moreover, the attractive PK properties of the series were maintained by inclusion of the methyl ester, allowing us, in effect, to reduce the half-life (). The corresponding Picroside II was found to be the main metabolite in human microsomes and it was 100-fold less active against hDHODH than the ester.
In an attempt to identify the specific non-covalent binding interactions which were responsible for the high binding affinity, co-crystal structures with the human enzyme were determined for selected analogs. Herein we describe the structure of DHODH complex with the inhibitor . The structure was solved at a resolution of 2.18Å, and revealed the detailed binding mode of the ligand bound to the putative ubiquinone binding site.
The ligand forms two specific hydrogen bonds to DHODH, namely to the side chain residues Arg136 and Tyr356. Furthermore, binding of ligand is enhanced by a variety of hydrophobic interactions involving residues of the N-terminal helices α1 and α2—namely Tyr38, Met43, Leu46, Pro52, His56, Ala59, Phe62, Leu68 as well as Phe98, Met111, Thr360 and Pro364 ().
These hydrophobic interactions are consistent with the good activity of the lipophilic biphenyl derivatives (compounds –)—in particular, compounds , , , , , and with an IC <50nM.
The co-crystal structure reveals that the newly introduced phenyl group fills the above hydrophobic binding cavity in a more complementary fashion than the CF of ().
Inhibition of DHODH at a cellular level translates into the inhibition of proliferation of mitogen-stimulated human peripheral blood mononuclear cells (hPBMCs), which can be measured by incorporation of tritiated thymidine following 72h of incubation of cells with phytohemagglutinin (PHA). Data for few selected compounds are presented in . Thus, enzymatic inhibition of hDHODH was translated into a good cellular effect, in particular compounds and which displayed potent antiproliferative activities on PBMCs.
The in vivo anti-inflammatory effect of compound was further completed using the functional model of adjuvant induced arthritis (AIA) in rats as shown in .
Compound was shown to be active in the AIA model, although with a lower potency than teriflunomide. These results are not surprising in the light of the reduced half-life and the lower potency as rat DHODH inhibitor of compound versus teriflunomide. Given that compound is 10 times more potent in the human enzyme, we would expect a similar efficacy as teriflunomide in humans and with a more acceptable pharmacokinetic profile.
A new family of potent and orally bioavailable hDHODH inhibitors has been designed, synthesized and characterized. Compound is more than 20-fold more potent than teriflunomide as an anti-proliferative in PBMCs and has an attractive PK profile in rat which is likely to obviate the liabilities (a half life of 2weeks in man) of teriflunomide. Compound has also shown efficacy in the experimental model of arthritis, AIA. Thus, compound has been identified as an attractive starting point for further optimization. Further studies have addressed replacements for the β-hydroxyenamide moiety, to minimize the possible biphenylaniline metabolite that could be formed during Phase I metabolism. This work will be reported in due course.
The authors wish to thank Proteros Biostructures GmbH for X-ray structure determination of human DHODH in complex with compound 10 (PDB code: 3U2O).
Introduction Theileria equi and Babesia caballi, are pear-shaped erythrocytic protozoan parasites that belong to the families Theileriidae and Babesidae, members of the phylum Apicomplexa (OIE, 2008). These two parasites cause acute haemolytic anemia in horses, mules, donkeys and zebras, known as equine piroplasmosis (EP) (Rothschild, 2013). T. equi and B. caballi have many common tick vectors and frequently co-infect horses. Both are found globally where tick vectors are present, and are endemic to most tropical and subtropical areas as well as temperate climatic zones, including many parts of Europe, Africa, Arabia and Asia (except Japan) (Brünning, 1996). Infected horses remain seropositive for a period of several years (B. caballi) to a lifetime (B. caballi and T. equi) (Rothschild, 2013). However, active infection results in acute cases and death, which has a significant impact on the international movement of horses (Knowles, 1996). Economic losses associated with EP are significant and include the cost of treatment, especially in acutely infected horses, abortion in the last trimester of gestation, loss of performance, death, and restrictions in meeting international requirements related to exportation or participation in equestrian sporting events (DE Waal, 1992, Kerber et al., 1999, Lewis et al., 1999). T. equi reported resistant to babecidal drugs (Brünning, 1996), while diminazene aceturate treatment in horses and mules results in mild to severe toxicity (Tuntasuvan et al., 2003). Therefore, an alternative chemotherapeutic drug is needed for control of EP. Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine biosynthesis pathway, essential for survival of apicomplexan parasites by providing UMP, which is essential for RNA and DNA synthesis (Shambaugh, 1979, Phillips et al., 2008). DHODH inhibitors have been studied for treatment of various pathogens. For example, redoxal, dichloroallyl lawsone (DCL), and three analogs of A77 1726, DHODH inhibitors were tested with Plasmodium falciparum (Baldwin et al., 2002). The A77-1726, leflunomide, MD249 and MD209 compounds were evaluated on the inhibition of Toxoplasma gondii DHODH (TgDHODH) (Hortua Triana et al., 2012). Leflunomide has been studied as a drug for treatment of arthritis (McRobert and McConkey, 2002). The quinone co-substrate of the dihydroorotate dehydrogenase, atovaquone, is active against Plasmodium, Toxoplasma gondii (Baggish and Hill, 2002), Babesia spp. (Weiss, 2002) and Babesia gibsoni (Matsuu et al., 2004). Atovaquone was verified to be used for the therapeutic window (efficacy versus toxicity), as it was an uncompetitive inhibitor of human DHODH and a non-competitive inhibitor of the rat DHODH (Knecht et al., 2000). Brequinar derivatives were also proved as potentially effective inhibitors against P. falciparum DHODH (Hurt et al., 2006). Our previous study showed that DHODH inhibitors effectively inhibited the activity of rBboDHODH as well as the growth of Babesia bovis in vitro. (Kamyingkird et al., 2014). However, currently there is no literature evaluating DHODH inhibitors as novel chemotherapeutic drug for EP. In the present study, we compared T. equi DHODH to other apicomplexan protozoa DHODHs using bioinformatics and immunoblot analysis. In addtion, DHODH inhibitors were evaluated for inhibition of T. equi and B. caballi by in vitro inhibition assay.