In conclusion we have discovered two new derivatives and
In conclusion, we have discovered two new derivatives ( and ) that are potent inhibitors of DHODH. H and C NMR spectroscopic data revealed that these compounds undergo ready isomerisation at room temperature in -DMSO, but the docking studies indicate that there is neither conformation nor configuration which binds preferentially to DHODH. This flexibility is favourable for inhibitors of this channel that require extensive positioning to reach their binding site. X-ray crystallographic study of the red crystal form of ()- reveals shortened N2C15 and lengthened C15C16 bonds. The delocalisation of the charge onto the nitro group must promote the hydrogen bonding interactions between the nitro group and the N9H, but also with any H-bond donor group on the enzyme. This is consistent with the observations made by Phillips and co-workers where an intrinsic dipole contribution in their triazolopyrimidine class of compounds is also believed to add to the hydrogen bonding and thus binding of the inhibitors to the enzyme. Overall, these results further add to the knowledge of inhibitor binding to the co-factor binding site of DHODH and may assist in the design and optimisation of other, more drug-like inhibitors of dihydroorotate dehydrogenase.
We thank the University of Hull for funding, BBSRC for support (GM), and the EPSRC National Mass Spectrometry Service (Swansea) for accurate mass determination of 2e and 3e.
Introduction Mitochondria are vital organelles for most eukaryotic glycogen phosphorylase (Karnkowska et al., 2016). They carry their own DNA (mtDNA) and are involved in a number of essential processes. The signature feature of mitochondria is oxidative phosphorylation (OXPHOS), responsible for respiration and ATP formation. Respiration is performed by four respiratory complexes (RCs; i.e., CI-IV) that associate into supercomplexes (SCs) and generate a proton gradient across the inner mitochondrial membrane (IMM) that is used by ATP synthase (CV) to produce ATP (Acin-Perez et al., 2008, Althoff et al., 2011, Moreno-Lastres et al., 2012, Gu et al., 2016, Letts et al., 2016, Wu et al., 2016). Respiration also drives biosynthetic pathways directly or via the tricarboxylic acid cycle (Bezawork-Geleta et al., 2018). Essential protein subunits of OXPHOS complexes are encoded by nuclear DNA and mtDNA. Therefore, when mtDNA is absent or damaged, OXPHOS is severely compromised (Brandon et al., 2006, Wallace, 2012). Recently we showed that cancer cells deficient in OXPHOS due to mtDNA depletion (ρ0 cells) cannot form tumors unless they acquire functional mtDNA from host stroma (Tan et al., 2015) by transfer of whole mitochondria (Dong et al., 2017). Other researchers support our findings (Osswald et al., 2015, Lei and Spradling, 2016, Moschoi et al., 2016, Strakova et al., 2016). These observations suggest that functional OXPHOS is essential for tumorigenesis, a concept consistent with other reports (LeBleu et al., 2014, Viale et al., 2014). Furthermore, they conform to the notion that the Warburg effect is associated with altered biosynthetic needs of cancer cells rather than with cancer-linked mitochondrial damage (Vander Heiden et al., 2009, Vander Heiden and DeBerardinis, 2017). However, important questions remain unresolved. Foremost, it is unclear which aspect of OXPHOS activity is limiting for tumor growth. ATP production is the best known function of OXPHOS, but proliferating cells also require respiration for its oxidizing power and to produce aspartate for pyrimidine biosynthesis (Birsoy et al., 2015, Sullivan et al., 2015, Titov et al., 2016). Further, OXPHOS directly drives the respiration-coupled mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) that converts dihydroorotate (DHO) to orotate in the de novo pyrimidine synthesis pathway (Loffler et al., 2005). Here we analyzed temporal events preceding tumor formation in ρ0 cancer cells in the context of horizontal transfer of mtDNA in vivo and linked this to genetic manipulations of the OXPHOS system. Our results indicate that a key event facilitating tumor growth upon respiration recovery is reactivation of DHODH-driven pyrimidine synthesis.