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  • The current study provides evidence for a

    2020-02-26

    The current study provides evidence for a previously unknown role for DHODH as a growth/survival factor in KRAS mutant tumors. While many aspects of the link between DHODH inhibition and survival of KRAS mutant cells remain to be established, de novo pyrimidine biosynthesis appears to be a crucial pathway for KRAS mutant cells. The connection between KRAS mutant status and a requirement for DHODH enzymatic activity may enable new therapeutic approaches for KRAS mutant cancers.
    Significance
    STAR★Methods
    Acknowledgments We thank Dr. Laura Shelton (Human Metabolome Technologies) for helpful discussions. We thank Dr. Dietmar Hoffmann and the Sanofi Molecular Cloning group for generation of inducible shRNA vectors for KRAS knockdown. We thank Dr. Joern Hopke for microscopic image acquisition and analysis.
    Introduction Poly (ADP-ribose) polymerases (PARPs) comprise 18 putative family members of the nuclear enzymes that have significant roles in multifunctional cellular processes, including detection and repair of damaged DNA and RNA. PARP-1, PARP-2 and PARP-3 are the best studied members of this family of enzymes due to their role in DNA repair. Other distinct biochemical activities of PARP-1 are epigenetic chromatic modifications, genomic stability regulations,7, 22 replication and transcription of DNA and distinctive cell death formation known as parthanatos.10, 14 PARP-1 is known to be the trigger point in the DNA repair mechanism for single strand breaks where it acts as the DNA damage-sensing enzyme. In response to DNA damage that may have occurred due to radiation or chemotherapeutic agents, PARP-1 initiates its repair process by binding to the damaged site and catalyzing the synthesis of long, branched poly (ADP-ribose) chains using nicotinamide T7 High Yield Cy3 RNA dinucleotide (NAD+) as the substrate. These actions of PARP-1 result in the resistance that frequently develops after cancer therapy. Hence inhibition of the PARP-1 enzyme is believed to enhance sensitivity towards radiotherapy and certain kinds of DNA targeting cancer chemotherapies. To date, a significant number of potent PARP-1 inhibitors have been reported accentuating the role of PARP-1. Inhibition of PARP in homologous recombination (HR) deficient tumor cells have also exclusively explained the crucial role of PARP-1 in DNA repair.11, 28 These inhibitors generally bind to the nicotinamide binding site of the PARP-1 catalytic domain, thus inhibiting automodification and subsequent release of the enzyme from the site of DNA damage as well as preventing the access of other repair proteins to the site of DNA damage. The binding of these inhibitors mimics the binding mode of nicotinamide towards PARP-1 with key interactions to Ser243 (CO to OH Ser) and Gly202 (CO to NH Gly and NH to CO Gly) through hydrogen-bonding and π–π stacking with Tyr246, which is approximately coplanar with the benzimidazole moiety of the ligand. In addition, Griffin and co-workers reported an intramolecular hydrogen bond between the carboxamide hydrogen on C2 of the indole ring to the nitrogen in the indole ring. This intramolecular H-bond resulted in a pseudo-6-membered ring, creating a rigid 6:6:5 tricyclic system which improved its potency.15, 25 A water molecule also plays a crucial role at the active site by interacting with the catalytically important carboxylate group of Glu327 which forms a hydrogen bond with the NH indole of benzimidazole of the ligand.29, 30 Inhibition of pyrimidine biosynthesis has been shown to have an efficacious anti-proliferative effect on cells that are dividing rapidly. Mitochondrial enzyme, dihydroorotate dehydrogenase (DHODH) catalyzes the fourth step in the de novo biosynthetic pathway of pyrimidines, converting dihydroorotate to orotate by oxidative reaction, with flavin mononucleotide (FMN) and ubiquinone (CoQ) acting as co-factors. This enzyme has been identified as a therapeutic target for treatment of cancer,9, 12 as well as several autoimmune disorders such as rheumatoid arthritis and multiple sclerosis. Inhibition of enzymatic activities has been reported on hDHODH and PfDHODH by X-ray crystallographic studies with known inhibitors such as leflunomide, teriflunomide (the active metabolite of leflunomide) and brequinar. These inhibitors are positioned in the suggested ubiquinone binding site where polar and hydrophobic residues contribute to the binding. The carboxylic acid group from the inhibitors shows good hydrogen-bonding interactions with the guanidinyl moiety of Arg136 and an additional hydrogen bonding interaction to the side chain of Gln47.