Hydroxyazole scaffold-based Plasmodium falciparum dihydroorotate dehydrogenase inhibitors: Synthesis, biological evaluation and X-ray structural studies.

Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) has been clinically validated as a target for antimalarial drug discovery, as a triazolopyrimidine class inhibitor (DSM265) is currently undergoing clinical development. Here, we have identified new hydroxyazole scaffold-based PfDHODH inhibitors belonging to two different chemical series. The first series was designed by a scaffold hopping strategy that exploits the use of hydroxylated azoles. Within this series, the hydroxythiadiazole 3 was identified as the best selective PfDHODH inhibitor (IC50 12.0 µM). The second series was designed by modulating four different positions of the hydroxypyrazole scaffold. In particular, hydroxypyrazoles 7e and 7f were shown to be active in the low µM range (IC50 2.8 and 5.3 µM, respectively). All three compounds, 3, 7e and 7f showed clear selectivity over human DHODH (IC50 > 200 µM), low cytotoxicity, and retained micromolar activity in P. falciparum-infected erythrocytes. The crystallographic structures of PfDHODH in complex with compounds 3 and 7e proved their binding mode, supplying essential data for future optimization of these scaffolds.

The structures of human dihydroorotate dehydrogenase with and without inhibitor reveal conformational flexibility in the inhibitor and substrate binding sites.

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.

Synthesis and biological evaluation of new 1,2-dihydro-4-hydroxy-2-oxo-3-quinolinecarboxamides for treatment of autoimmune disorders: structure-activity relationship.

Roquinimex-related 3-quinolinecarboxamide derivatives were prepared and evaluated for treatment of autoimmune disorders. The compounds were tested in mice for their inhibitory effects on disease development in the acute experimental autoimmune encephalomyelitis model and selected compounds in the beagle dog for induction of proinflammatory reaction. Structure-activity relationships are discussed. Compound 8c, laquinimod, showed improved potency and superior toxicological profile compared to the lead compound roquinimex (1b, Linomide) and was selected for clinical studies (currently in phase II).

An N-(alkylcarbonyl)anthranilic acid derivative prolongs cardiac allograft survival synergistically with cyclosporine A in a high-responder rat model.

We investigated the immunosuppressive effects of the dihydroortate dehydrogenase (DHODH) inhibitor compounds ABR-222417 and ABR-224050 from Active Biotech (Sweden). We verified the inhibitory effects of these compounds on the proliferation of CD4(+) and CD8(+) T-cells in vivo by using superantigen staphylococcus enterotoxin A (SEA)-mediated proliferation test. To evaluate their efficacy, the compounds were screened in a low-responder heart allograft transplantation model in rats [heart from Piebald Virol Glaxo (PVG) transplanted to Dark Agouti (DA)]. The immunosuppressive effects of the compounds were then investigated in a high-responder model (DA to PVG). Treatment with ABR-222417 (30 mg/kg) was more efficient than that with ABR-224050 (10 mg/kg), and the former provided a longer graft median survival time (MST, 29.5 days) than the latter (MST, 18.5 days). Furthermore, there was a marked increase in graft survival time (53 days) when low doses of ABR-222417 and cyclosporine A (CsA) were used in combination. No sign of tolerability problems was detected using this combination or when ABR-222417 was used singly at a higher dose. Furthermore, T-cell proliferation studies in vitro support that the anti proliferative effect of ABR-222417 is caused by inhibition of de novo pyrimidine synthesis, which is the consequence of DHODH inhibition. These results show that ABR-222417 had marked immunosuppressive effects on the heart allograft transplantation and that it exerts an even more powerful inhibitory effect on graft rejection when used in combination with CsA, with good tolerability.

Inhibition of human DHODH by 4-hydroxycoumarins, fenamic acids, and N-(alkylcarbonyl)anthranilic acids identified by structure-guided fragment selection.

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.