ABSTRACT
S100A4 and S100A9 proteins have been described as playing roles in the control of tumor growth and metastasis. We show here that a chemical probe, oxyclozanide (OX), selected for inhibiting the interaction between S100A9 and the receptor for advanced glycation end-products (RAGE) interacts with both S100A9 and S100A4. Furthermore, we show that S100A9 and S100A4 interact with RAGE and TLR4; interactions that can be inhibited by OX. Hence, S100A4 and S100A9 display similar functional elements despite their primary sequence diversity. This was further confirmed by showing that S100A4 and S100A9 dimerize both in vitro and in vivo. All of these interactions required levels of Zn++ that are found in the extracellular space but not intracellularly. Interestingly, S100A4 and S100A9 are expressed by distinct CD11b+ subpopulations both in healthy animals and in animals with either inflammatory disease or tumor burden. The functions of S100A9 and S100A4 described in this paper, including heterodimerization, may therefore reflect S100A9 and S100A4 that are released into the extra-cellular milieu.
Subject(s)
Calgranulin B/metabolism , Encephalomyelitis, Autoimmune, Experimental/metabolism , Lymphoma/metabolism , Molecular Probes/metabolism , Oxyclozanide/metabolism , S100 Proteins/metabolism , Animals , Blotting, Western , CD11b Antigen/metabolism , Calgranulin B/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dimerization , Extracellular Fluid/metabolism , Gene Expression Regulation/physiology , Humans , Mice , Mice, Inbred C57BL , Molecular Structure , Oxyclozanide/pharmacology , S100 Calcium-Binding Protein A4 , S100 Proteins/chemistry , Toll-Like Receptor 4/metabolism , Zinc/metabolismABSTRACT
A strategy that combines virtual screening and structure-guided selection of fragments was used to identify three unexplored classes of human DHODH inhibitor compounds: 4-hydroxycoumarins, fenamic acids, and N-(alkylcarbonyl)anthranilic acids. Structure-guided selection of fragments targeting the inner subsite of the DHODH ubiquinone binding site made these findings possible with screening of fewer than 300 fragments in a DHODH assay. Fragments from the three inhibitor classes identified were subsequently chemically expanded to target an additional subsite of hydrophobic character. All three classes were found to exhibit distinct structure-activity relationships upon expansion. The novel N-(alkylcarbonyl)anthranilic acid class shows the most promising potency against human DHODH, with IC(50) values in the low nanomolar range. The structure of human DHODH in complex with an inhibitor of this class is presented.
Subject(s)
4-Hydroxycoumarins/chemistry , Enzyme Inhibitors/chemistry , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , ortho-Aminobenzoates/chemistry , 4-Hydroxycoumarins/chemical synthesis , 4-Hydroxycoumarins/pharmacology , Binding Sites , Computer Simulation , Dihydroorotate Dehydrogenase , Drug Discovery , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Humans , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Structure-Activity Relationship , ortho-Aminobenzoates/chemical synthesis , ortho-Aminobenzoates/pharmacologyABSTRACT
Inhibitors of dihydroorotate dehydrogenase (DHODH) have been suggested for the treatment of rheumatoid arthritis, psoriasis, autoimmune diseases, Plasmodium, and bacterial and fungal infections. Here we present the structures of N-terminally truncated (residues Met30-Arg396) DHODH in complex with two inhibitors: a brequinar analogue (6) and a novel inhibitor (a fenamic acid derivative) (7), as well as the first structure of the enzyme to be characterized without any bound inhibitor. It is shown that 7 uses the "standard" brequinar binding mode and, in addition, interacts with Tyr356, a residue conserved in most class 2 DHODH proteins. Compared to the inhibitor-free structure, some of the amino acid side chains in the tunnel in which brequinar binds and which was suggested to be the binding site of ubiquinone undergo changes in conformation upon inhibitor binding. Using our data, the loop regions of residues Leu68-Arg72 and Asn212-Leu224, which were disordered in previously studied human DHODH structures, could be built into the electron density. The first of these loops, which is located at the entrance to the inhibitor-binding pocket, shows different conformations in the three structures, suggesting that it may interfere with inhibitor/cofactor binding. The second loop has been suggested to control the access of dihydroorotate to the active site of the enzyme and may be an important player in the enzymatic reaction. These observations provide new insights into the dynamic features of the DHODH reaction and suggest new approaches to the design of inhibitors against DHODH.
Subject(s)
Enzyme Inhibitors/metabolism , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Binding Sites/genetics , Biphenyl Compounds/chemistry , Biphenyl Compounds/metabolism , Carboxylic Acids/chemistry , Carboxylic Acids/metabolism , Crystallography, X-Ray , Dihydroorotate Dehydrogenase , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Flavin Mononucleotide/metabolism , Humans , Naphthalenes/chemistry , Naphthalenes/metabolism , Orotic Acid/analogs & derivatives , Orotic Acid/chemistry , Orotic Acid/metabolism , Oxidoreductases Acting on CH-CH Group Donors/chemistry , Oxidoreductases Acting on CH-CH Group Donors/genetics , Protein Binding , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ubiquinone/metabolism , ortho-Aminobenzoates/chemical synthesis , ortho-Aminobenzoates/chemistry , ortho-Aminobenzoates/metabolismSubject(s)
Galectins/antagonists & inhibitors , Amino Sugars/chemical synthesis , Amino Sugars/chemistry , Amino Sugars/pharmacology , Animals , Binding Sites , Drug Design , Enzyme-Linked Immunosorbent Assay , Fluorescence Polarization , Fluorescent Dyes/chemical synthesis , Galectins/chemistry , In Vitro Techniques , Models, Molecular , Molecular StructureABSTRACT
Nitration of some 2-substituted pyrimidine-4,6-diones in sulfuric acid was studied, which afforded previously unknown 5,5-gem-dinitropyrimidine-4,6-diones in high yields. The gem-dinitro products were easily attacked by nucleophiles with concomitant formation of gem-dinitroacetyl derivatives, which in turn could be further hydrolyzed to salts of dinitromethane and triureas.
