Inhibition of p70 S6 Kinase (S6K1) Activity by A77 1726 and Its Effect on Cell Proliferation and Cell Cycle Progress
Michelle E. DoscasAshley J. WilliamsonLydia UshaYedida BogachkovGeetha RaoFei XiaoYimin WangCarl E. RubyHoward L. KaufmanJingsong ZhouJames W. WilliamsYi LiXiulong Xu
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Leflunomide is a novel immunomodulatory drug prescribed for treating rheumatoid arthritis.It inhibits the activity of protein tyrosine kinases and dihydroorotate dehydrogenase, a rate-limiting enzyme in the pyrimidine nucleotide synthesis pathway.Here, we report that A77 1726, the active metabolite of leflunomide, inhibited the phosphorylation of ribosomal protein S6 and two other substrates of S6K1, insulin receptor substrate-1 and carbamoyl phosphate synthetase 2, in an A375 melanoma cell line.A77 1726 increased the phosphorylation of AKT, p70 S6 (S6K1), ERK1/2, and MEK through the feedback activation of the IGF-1 receptor-mediated signaling pathway.In vitro kinase assay revealed that leflunomide and A77 1726 inhibited S6K1 activity with IC 50 values of approximately 55 and 80 μM, respectively.Exogenous uridine partially blocked A77 1726-induced inhibition of A375 cell proliferation.S6K1 knockdown led to the inhibition of A375 cell proliferation but did not potentiate the antiproliferative effect of A77 1726.A77 1726 stimulated bromodeoxyuridine incorporation in A375 cells but arrested the cell cycle in the S phase, which was reversed by addition of exogenous uridine or by MAP kinase pathway inhibitors but not by rapamycin and LY294002 (a phosphoinositide 3-kinase inhibitor).These observations suggest that A77 1726 accelerates cell cycle entry into the S phase through MAP kinase activation and that pyrimidine nucleotide depletion halts the completion of the cell cycle.Our study identified a novel molecular target of A77 1726 and showed that the inhibition of S6K1 activity was in part responsible for its antiproliferative activity.Our study also provides a novel mechanistic insight into A77 1726-induced cell cycle arrest in the S phase.Keywords:
Dihydroorotate Dehydrogenase
A protein with high affinity (Kd 12 nM) for the immunomodulatory compound A77 1726 has been isolated from mouse spleen and identified as the mitochondrial enzyme dihydroorotate dehydrogenase (EC 1.3.3.1). The purified protein had a pI 9.6-9.8 and a subunit Mr of 43,000. Peptides derived from the mouse protein displayed high microsequence similarity to human and rat dihydroorotate dehydrogenase with, respectively, 35 and 39 out of 43 identified amino acids identical. Dihydroorotate dehydrogenase catalyzes the fourth step in de novo pyrimidine biosynthesis. The in vitro antiproliferative effects of A77 1726 are mediated by enzyme inhibition and can be overcome by addition of exogenous uridine. The rank order of potency of A77 1726 and its analogues in binding or enzyme inhibition was similar to that for inhibition of the mouse delayed type hypersensitivity response. It is proposed that inhibition of dihydroorotate dehydrogenase is an in vivo mechanism of action of the A77 1726 class of compounds. This was confirmed using uridine to counteract inhibition of the murine acute graft versus host response. A protein with high affinity (Kd 12 nM) for the immunomodulatory compound A77 1726 has been isolated from mouse spleen and identified as the mitochondrial enzyme dihydroorotate dehydrogenase (EC 1.3.3.1). The purified protein had a pI 9.6-9.8 and a subunit Mr of 43,000. Peptides derived from the mouse protein displayed high microsequence similarity to human and rat dihydroorotate dehydrogenase with, respectively, 35 and 39 out of 43 identified amino acids identical. Dihydroorotate dehydrogenase catalyzes the fourth step in de novo pyrimidine biosynthesis. The in vitro antiproliferative effects of A77 1726 are mediated by enzyme inhibition and can be overcome by addition of exogenous uridine. The rank order of potency of A77 1726 and its analogues in binding or enzyme inhibition was similar to that for inhibition of the mouse delayed type hypersensitivity response. It is proposed that inhibition of dihydroorotate dehydrogenase is an in vivo mechanism of action of the A77 1726 class of compounds. This was confirmed using uridine to counteract inhibition of the murine acute graft versus host response.
Dihydroorotate Dehydrogenase
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Dihydroorotate Dehydrogenase
Pyrimidine metabolism
Teriflunomide
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Dihydroorotate dehydrogenase (DHODH), a novel and recently discovered enzyme, is involved in the biosynthesis of uridine. Leflunomide (CAS 75706-12-6), a drug approved for the treatment of treat rheumatoid arthritis (RA), was identified as an inhibitor of DHODH. Structure based drug design using the leflunomide/DHODH X-ray structure yielded novel inhibitors with improved pharmacological properties. Such drug candidates are in clinical trials against various autoimmune diseases.
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Pyrimidine metabolism
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Proper nucleosides availability is crucial for the proliferation of living entities (eukaryotic cells, parasites, bacteria, and virus). Accordingly, the uses of inhibitors of the de novo nucleosides biosynthetic pathways have been investigated in the past. In the following we have focused on dihydroorotate dehydrogenase (DHODH), the fourth enzyme in the de novo pyrimidine nucleosides biosynthetic pathway. We first described the different types of enzyme in terms of sequence, structure, and biochemistry, including the reported bioassays. In a second part, the series of inhibitors of this enzyme along with a description of their potential or actual uses were reviewed. These inhibitors are indeed used in medicine to treat autoimmune diseases such as rheumatoid arthritis or multiple sclerosis (leflunomide and teriflunomide) and have been investigated in treatments of cancer, virus, and parasite infections (i.e., malaria) as well as in crop science.
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The inhibition of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) potentially represents a new treatment option for malaria, as P. falciparum relies entirely on a de novo pyrimidine biosynthetic pathway for survival. Herein, we report a series of pyrimidone derivatives as novel inhibitors of PfDHODH. The most potent compound, 26, showed high inhibition activity against PfDHODH (IC50 = 23 nM), with >400-fold species selectivity over human dihydroorotate dehydrogenase (hDHODH). The brand-new inhibitor scaffold targeting PfDHODH reported in this work may lead to the discovery of new antimalarial agents.
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Pyrimidine metabolism
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Dihydroorotate Dehydrogenase
Pyrimidine metabolism
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Human dihydroorotate dehydrogenase ( DHODH ), the enzyme that catalyzes the rate‐limiting step in de novo pyrimidine biosynthesis, is considered to be an attractive target for potential treatment of autoimmune disease and cancer. Here, we present a novel class of human DHODH inhibitors with high inhibitory potency. The high‐resolution crystal structures of human DHODH complexed with various agents reveal the details of their interactions. Comparisons with the binding modes of teriflunomide and brequinar provide insights that may facilitate the development of new inhibitors targeting human DHODH .
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Pyrimidine biosynthesis presents an attractive drug target in malaria parasites due to the absence of a pyrimidine salvage pathway. A set of compounds designed to inhibit the Plasmodium falciparum pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (PfDHODH) was synthesized. PfDHODH-specific inhibitors with low nanomolar binding affinities were identified that bind in the N-terminal hydrophobic channel of dihydroorotate dehydrogenase, the presumed site of ubiquinone binding during oxidation of dihydroorotate to orotate. These compounds also prevented growth of cultured parasites at low micromolar concentrations. Models that suggest the mode of inhibitor binding is based on shape complementarity, matching hydrophobic regions of inhibitor and enzyme, and interaction of inhibitors with amino acid residues F188, H185, and R265 are supported by mutagenesis data. These results further highlight PfDHODH as a promising new target for chemotherapeutic intervention in prevention of malaria and provide better understanding of the factors that determine specificity over human dihydroorotate dehydrogenase.
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Malaria is a severe human disease and a global health problem because of drug-resistant strains. Drugs reported to prevent the growth of Plasmodium parasites target various phases of the parasites' life cycle. Antimalarial drugs can inhibit key enzymes that are responsible for the cellular growth and development of parasites. Plasmodium falciparum dihydroorotate dehydrogenase is one such enzyme that is necessary for de novo pyrimidine biosynthesis. This review focuses on various medicinal chemistry approaches used for the discovery and identification of selective P. falciparum dihydroorotate dehydrogenase inhibitors as antimalarial agents. This comprehensive review discusses recent advances in the selective therapeutic activity of distinct chemical classes of compounds as P. falciparum dihydroorotate dehydrogenase inhibitors and antimalarial drugs.
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Pyrimidine metabolism
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Dihydroorotate Dehydrogenase
Pyrimidine metabolism
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